Inhibition of oxidative stress in atrial fibrillation

ABSTRACT

Disclosed herein are pharmaceutical compositions and methods for inhibiting oxidative stress in a subject having atrial or ventricular arrhythmias, ventricular failure or heart failure. The methods include administering an effective amount of a NOX2 inhibitor agent to the subject, wherein said administering is under conditions such that a level of oxidative stress in myocardial tissue is reduced or eliminated. The pharmaceutical compositions include a NOX2 inhibitor agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. non-provisional patentapplication Ser. No. 15/943,069, now U.S. Pat. No. 10,988,767, issuedApr. 27, 2021, which is a divisional of U.S. non-provisional patentapplication Ser. No. 14/542,501, now U.S. Pat. No. 9,932,588, issuedApr. 3, 2018, which claims benefit of priority under 35 U.S.C. 119 toU.S. provisional patent application Ser. No. 61/904,925, filed Nov. 15,2013, each of which are herein incorporated by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSOREDRESEARCH OR DEVELOPMENT

This invention was made with government support under HL093490 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF INVENTION

The present disclosure relates to compositions and methods forpreventing certain cardiac damage. In particular, compositions andmethods of inhibiting oxidative stress in a subject suffering fromatrial fibrillation is provided.

BACKGROUND

Atrial Fibrillation (AF) is the most common heart rhythm disorder(Benjamin E J, Levy D, Vaziri S M, D'Agostino R B, Belanger A J, Wolf PA. “Independent risk factors for atrial fibrillation in apopulation-based cohort. The Framingham Heart Study,” JAMA 1994;271:840-4), and is a major risk factor for stroke and HF(Balasubramaniam R, Kistler P M. AF and “Heart failure: the chicken orthe egg?” Heart 2009; 95:535-9; Lakshminarayan K, Anderson D C, Herzog CA, Qureshi A I. “Clinical epidemiology of atrial fibrillation andrelated cerebrovascular events in the United States,” Neurologist 2008;14:143-50; Lip G Y, Kakar P, Watson T. “Atrial fibrillation—the growingepidemic” [comment], Heart 2007; 93:542-3). Since a majority of AFtriggers arise in the pulmonary veins (PVs) and the adjoining adjoiningposterior left atrium (PLA), ablation procedures that electricallyisolate the PVs have emerged in recent years as a viable therapy forfocal AF. Nonetheless, moderately high ablation success rates have onlybeen achieved in selected patients (Nademanee K, McKenzie J, Kosar E etal. “A new approach for catheter ablation of atrial fibrillation:mapping of the electrophysiologic substrate” [see comment], J. Am. Coll.Cardiol. 2004; 43:2044-53; Nademanee K, Schwab M C, Kosar E M et al“Clinical outcomes of catheter substrate ablation for high-risk patientswith atrial fibrillation,” J. Am. Coll. Cardiol. 2008; 51:843-9; TaylorG W, Kay G N, Zheng X, Bishop S, Ideker R E. “Pathological effects ofextensive radiofrequency energy applications in the pulmonary veins indogs,” Circulation 2000; 101:1736-42). Indeed, in patients withstructural heart disease e.g. heart failure (HF), success rates do notexceed 50-60% (Estner H L, Hessling G, Ndrepepa G et al.“Electrogram-guided substrate ablation with or without pulmonary veinisolation in patients with persistent atrial fibrillation,” Europace2008; 10:1281-7; Weerasooriya R, Khairy P, Litalien J et al. “Catheterablation for atrial fibrillation: are results maintained at 5 years offollow-up?” J. Am. Coll. Cardiol. 2011; 57:160-6). One reason for thislow efficacy is that current ablation strategies primarily employ ananatomical, ‘one-size fits all’ strategy (with some minor variations)that does not address the specific mechanisms underlying AF (BenMorrison T, Jared Bunch T, Gersh B J. “Pathophysiology of concomitantatrial fibrillation and heart failure: implications for management,”Nat. Clin. Pract. Cardiovasc. Med 2009; 6:46-56). Recent research hastherefore attempted to better define the mechanisms underlying AF, inorder to improve upon the success of ablation and to develop newbiological therapies for AF.

In the setting of structural heart disease—specifically HF—a variety ofmechanisms e.g. stretch, oxidative stress (OS), autonomic imbalance andstructural changes such as fibrosis are thought to contribute to avulnerable AF substrate (Nattel S. “From guidelines to bench:implications of unresolved clinical issues for basic investigations ofatrial fibrillation mechanisms,” Can. J. Cardiol. 2011; 27:19-26; NattelS, Burstein B, Dobrev D. “Atrial remodeling and atrial fibrillation:mechanisms and implications,” Circ. Arrhythm. Electrophysiol. 2008;1:62-73). OS is known to be elevated in the atria in AF (Youn J Y, ZhangJ, Zhang Yet at. “Oxidative stress in atrial fibrillation: an emergingrole of NADPH oxidase,” J. Mol. Cell. Cardiol. 2013; 62:72-9) andreactive oxygen species (ROS) have effects on the atrial actionpotential and Ca²⁺ cycling. However, the precise effects of ROS onatrial electrophysiology in the intact atria—and how theseelectrophysiological changes contribute to formation of AF substrate—arenot known.

Oxygen derivatives with instabilities and increased reactivity, e.g. O₂,H₂O₂, and OH, are generically termed ROS (Maejima Y, Kuroda J,Matsushima S, Ago T, Sadoshima J. “Regulation of myocardial growth anddeath by NADPH oxidase,” J. Mol. Cell. Cardiol. 2011; 50:408-16). WhileROS at low doses mediates physiological functions such as growth,differentiation, metabolism (id.), excess ROS damages DNA, protein andlipids, and causes cell death (id.). A wealth of research points toincreased OS as a key driver of cardiac remodeling caused by chronicpressure overload, loss of functional myocardium or AF (Kohlhaas M,Maack C. “Interplay of defective excitation-contraction coupling, energystarvation, and oxidative stress in heart failure,” Trends Cardiovasc.Med 2011; 21:69-73; Maulik S K, Kumar S. “Oxidative stress and cardiachypertrophy: a review,” Toxicol. Mech. Methods 2012; 22:359-66). Inaddition, chronic ROS elevation activates signaling pathways such asTGF-1, MAP kinase subfamilies (Hori M, Nishida K. “Oxidative stress andleft ventricular remodelling after myocardial infarction,” Cardiovas.Res. 2009; 81:457-64; Tsai K H, Wang W J, Lin C W et at. “NADPHoxidase-derived superoxide anion-induced apoptosis is mediated via theJNK-dependent activation of NF-kappaB in cardiomyocytes exposed to highglucose,” J. Cell. Physiol. 2012; 227:1347-57) that result in structuralchanges e.g. fibrosis. Moreover, ROS generation (e.g. by Ang II mediatedNOX activation) has been shown to lead to modifications of CaMKII(Erickson J R, He B J, Grumbach I M, Anderson I M. “CaMKII in thecardiovascular system: sensing redox states,” Physiol. Rev. 2011;91:889-915), an important serine-threonine kinase involved in a varietyof E-C coupling related processes in cardiac myocytes.

ROS are generated by the mitochondrial electron transport chain, thexanthine oxidase/dehydrogenase system, ‘uncoupled’ NOS, cytochrome P450and NADPH oxidases. The NADPH oxidase enzyme family are a major sourceof cardiovascular ROS (Murdoch C E, Zhang M, Cave A C, Shah A M. “NADPHoxidase-dependent redox signalling in cardiac hypertrophy, remodellingand failure,” Cardiovasc. Res. 2006; 71:208-15; Cave A C, Brewer A C,Narayanapanicker A et al. “NADPH oxidases in cardiovascular health anddisease,” Antioxid Redox. Signal. 2006; 8:691-728) with NOX2 being thedominant ROS-generating NADPH isoform in HF (Nabeebaccus A, Zhang M,Shah A M. “NADPH oxidases and cardiac remodeling,” Heart Fail. Rev.2011; 16:5-12; Maejima Y, Kuroda J, Matsushima S, Ago T, Sadoshima J.“Regulation of myocardial growth and death by NADPH oxidase,” J Mol.Cell. Cardiol 2011; Cave A C, Brewer A C, Narayanapanicker A et al.“NADPH oxidases in cardiovascular health and disease,” Antioxid Redox.Signal. 2006; 8:691-728; Zhang P, Hou M, Li Y et al “NADPH oxidasecontributes to coronary endothelial dysfunction in the failing heart,”Am. J. Physiol. Heart. Circ. Physiol. 2009; 296:H840-6; Dworakowski R,Alom-Ruiz S P, Shah A M. “NADPH oxidase-derived reactive oxygen speciesin the regulation of endothelial phenotype,” Pharmacol Rep. 2008;60:21-8). However, more recent studies indicate that NOX4 inmitochondria plays an essential role in mediating OS during pressureoverload-induced cardiac hypertrophy (Nabeebaccus A, Zhang M, Shah A M.“NADPH oxidases and cardiac remodeling,” Heart Fail. Rev. 2011; 16:5-12;Kuroda J, Ago T, Matsushima S, Zhai P, Schneider M D, Sadoshima J.“NADPH oxidase 4 (Nox4) is a major source of oxidative stress in thefailing heart,” Proc. Natl. Acad Sci. U.S.A. 2010; 107:15565-70; ZhangM, Brewer A C, Schroder K et al “NADPH oxidase-4 mediates protectionagainst chronic load-induced stress in mouse hearts by enhancingangiogenesis,” Proc. Natl. Acad Sci. U.S.A. 2010; 107:18121-6) NOX4 alsoappears to contribute significantly to the formation of fibrosis; inaddition, it appears to be activated by pro-fibrotic signaling pathwayse.g. TGF-β (Yeh Y H, Kuo C T, Chang G J, Qi X Y, Nattel S, Chen W J.“Nicotinamide adenine dinucleotide phosphate oxidase 4 mediates thedifferential responsiveness of atrial versus ventricular fibroblasts totransforming growth factor-beta,” Circ. Arrhythm. Electrophysiol 2013;6:790-8; Zhang M, Perino A, Ghigo A, Hirsch E, Shah A M. “NADPH oxidasesin heart failure: poachers or gamekeepers?” Antioxid Redox. Signal.2013; 18:1024-41).

Recent evidence indicates that OS also contributes to structural andelectrical remodeling in AF. Mihm et al demonstrated significantoxidative damage in atrial appendages of AF patients undergoing the Mazeprocedure (Huang C X, Liu Y, Xia W F, Tang Y H, Huang H. Oxidativestress: a possible pathogenesis of atrial fibrillation. Med Hypotheses2009; 72:466-7). Carnes et al. showed that dogs with sustained AF had anincrease in protein nitration, suggesting enhanced OS (Carnes C A,Janssen P M, Ruehr M L et at. “Atrial glutathione content, calciumcurrent, and contractility,” J. Biol. Chem. 2007; 282:28063-73; Carnes CA, Chung M K, Nakayama T et al “Ascorbate attenuates atrialpacing-induced peroxynitrite formation and electrical remodeling anddecreases the incidence of postoperative atrial fibrillation,” Circ.Res. 2001; 89:E32-8)

Kim et al. showed that NADPH oxidase (NOX2) was a major source of atrialROS in patients with AF (Kim Y M, Guzik T J, Zhang Y H et at. “Amyocardial Nox2 containing NAD(P)H oxidase contributes to oxidativestress in human atrial fibrillation,” Circ. Res. 2005; 97:629-36). Morerecently, Reilly et al. have shown that atrial sources of ROS vary withthe duration and substrate of AF, with NADPH oxidase being elevatedearly in AF (e.g. with post-operative AF) and with mitochondrialoxidases and uncoupled NOS being noted in long standing AF (Reilly S N,Jayaram R, Nahar K et al “Atrial sources of reactive oxygen species varywith the duration and substrate of atrial fibrillation: implications forthe antiarrhythmic effect of statins,” Circulation 2011; 124:1107-17).More recent data demonstrates that NOX4—which, as mentioned above,appears to contribute to the generation of mitochondrial ROS,specifically H₂O₂ (which more explicitly promotes fibrosis (Cucoranu I,Clempus R, Dikalova A et al. “NAD(P)H Oxidase 4 Mediates TransformingGrowth Factor-1-Induced Differentiation of Cardiac Fibroblasts IntoMyofibroblasts,” Circ. Res. 2005; 97:900-7))—is also elevated in AF(Joun et al. (2013) supra; Zhang J, Youn J Y, Kim A et al.“NOX4-dependent Hydrogen Peroxide Overproduction in Human AtrialFibrillation and HL-1 Atrial Cells: Relationship to Hypertension,”Front. Physiol. 2012; 3)

The detrimental electrical effects of an enhanced, pathological lateI_(Na) include the following: (i) diastolic depolarization during phase4 of the AP that may lead to spontaneous AP firing and abnormalautomaticity, (ii) an increase of AP duration, which may lead to EADsand triggered activity, as well as increased spatiotemporal differencesof repolarization time, which promote reentrant electrical activity; and(iii) the indirect effects of a late I_(Na)-induced increase of Na⁺entry to alter Ca²⁺ homeostasis in myocytes, which may lead to Ca²⁺alternans and DADs. Acquired conditions and drugs that enhance lateI_(Na) are associated with atrial tachyarrhythmias, ventriculartachyarrhythmias including torsades de pointes (TdP), afterpotentials(EADs, DADs), and triggered activity (Shryock J C, Song Y, Rajamani S,Antzelevitch C, Belardinelli L. “The arrhythmogenic consequences ofincreasing late I_(Na) in the cardiomyocyte,” Cardiovasc. Res. 2013;99:600-11). Recent investigations implicate a role for abnormal Ca²⁺handling in the genesis of ventricular and atrial arrhythmias (Aistrup GL, Balke C W, Wasserstrom J A. Arrhythmia triggers in heart failure: thesmoking gun of [Ca²⁺], dysregulation,” Heart Rhythm. 2011; 8:1804-8;Antoons G, Sipido K R “Targeting calcium handling in arrhythmias,”Europace 2008; 10:1364-9; Laurita K R, Rosenbaum D S. “Mechanisms andpotential therapeutic targets for ventricular arrhythmias associatedwith impaired cardiac calcium cycling,” J. Mol. Cell. Cardiol. 2008;44:31-43). Abnormal Ca²⁺ handling can contribute to arrhythmogenesisdirectly by triggering abnormal depolarizations and indirectly bymodulating action potential time course and duration. DADs are typicallyresult from cellular Ca²⁺ overload, with SCR increasing forward NCX andproducing an inward current resulting in DADs (Antoons et al. (2008)supra; Volders P G, Vos M A, Szabo B et at. “Progress in theunderstanding of cardiac early afterdepolarizations and torsades depointes: time to revise current concepts,” Cardiovasc. Res. 2000;46:376-92). More recent evidence has accumulated for Ca²⁺-mediated EADs(Volders et al. (2000) supra), which could contribute to triggeredactivity or at least prolong action potential duration. Indeed, abnormalCa²⁺ cycling and resulting Ca²⁺ transient alternans predisposes tochanges in the action potential that set up conditions for reentry(Laurita et al. (2008) supra). Abnormal SR Ca²⁺ release has also beensuggested to contribute to reentry in the intact atria, including thePVs (Chou C C, Nihei M, Zhou S et at. “Intracellular calcium dynamicsand anisotropic reentry in isolated canine pulmonary veins and leftatrium,” Circulation 2005; 111:2889-97). In HF, a number of atrialion-channel and E-C coupling proteins (Li D, Melnyk P, Feng J et at.“Effects of Experimental Heart Failure on Atrial Cellular and IonicElectrophysiology,” Circulation 2000; 101:2631-8

61. Yeh Y-H, Wakili R, Qi X-Y et at. “Calcium-Handling AbnormalitiesUnderlying Atrial Arrhythmogenesis and Contractile Dysfunction in DogsWith Congestive Heart Failure,” Circ. Arrhythm. Electrophysiol. 2008;1:93-102) can be modulated by ROS (Hool L C. “Reactive Oxygen Species inCardiac Signalling: From Mitochondria to Plasma Membrane Ion Channels,”Clin. Exp. Pharm. Phys. 2006; 33:146-51; Zima A V, Blatter L A. “Redoxregulation of cardiac calcium channels and transporters,” Cardiovasc.Res. 2006; 71:310-21; Nediani C, Raimondi L, Borchi E, Cerbai E. “NitricOxide/Reactive Oxygen Species Generation and Nitroso/Redox Imbalance inHeart Failure: From Molecular Mechanisms to Therapeutic Implications,”Antioxidants & Redox Signaling 2011; 14:289-331) at least in part viaROS activation of kinases and inactivation of phosphatases, resulting inaberrant phosphorylation (e.g. of RyR2 and phospholamban). Also, ROSdirectly decrease SERCA function, but increase NCX function (Kuster G M,Lancel S, Zhang J et at. “Redox-mediated reciprocal regulation of SERCAand Na⁺—Ca²⁺ exchanger contributes to sarcoplasmic reticulum Ca²⁺depletion in cardiac myocytes,” Free Rad Biol. Med 2010; 48:1182-7)which parallels the changes in SERCA and NCX in HF. Additionally, ROSincreases late/persistent I_(Na) (I_(Na_)late) (Luo A, Ma J, Zhang P,Zhou H, Wang W. “Sodium Channel Gating Modes During Redox Reaction,”Cell. Phys. Bioch 2007; 19:9-20), which again parallels that in HF(Valdivia C R, Chu W W, Pu J et al. “Increased late sodium current inmyocytes from a canine heart failure model and from failing humanheart,” J. Mol. Cell. Cardiol. 2005; 38:475-83) and I_(Na), late cansignificantly contribute to the induction of EADs and DADs (Li D, MelnykP, Feng J et al “Effects of Experimental Heart Failure on AtrialCellular and Ionic Electrophysiology,” Circulation 2000; 101:2631-8;Song Y, Shryock J C, Belardinelli L. “An increase of late sodium currentinduces delayed afterdepolarizations and sustained triggered activity inatrial myocytes,” Am. J. Physiol.-Heart and Circ. Physiol. 2008;294:H2031-H9; Wasserstrom J A, Sharma R, O'Toole M J et al “RanolazineAntagonizes the Effects of Increased Late Sodium Current onIntracellular Calcium Cycling in Rat Isolated Intact Heart,” J. Pharm.Exp. Ther. 2009; Undrovinas N, Maltsev V, Belardinelli L, Sabbah H,Undrovinas A. “Late sodium current contributes to diastolic cell Ca&lt;sup&gt;2+&lt;/sup&gt; accumulation in chronic heart failure,” J.Physiol. Sci. 2010; 60:245-57) both in ventricles and atria. Additionalpromotion of triggered activity could come from the increased Ca²⁺sensitivity of hyperphosphorylated RyR2s in HF (Terentyev D, Gyorke I,Belevych A E et al. “Redox Modification of Ryanodine ReceptorsContributes to Sarcoplasmic Reticulum Ca²⁺ Leak in Chronic HeartFailure,” Circ. Res. 2008; 103:1466-72), which together with OSmodifications of RyR2 (nitrosylation, oxidation) (Valdivia C R, Chu W W,Pu J et al “Increased late sodium current in myocytes from a canineheart failure model and from failing human heart,” J. Mol. Cell. Card2005; 38:475-83.

65. Song Y, Shryock J C, Belardinelli L. “An increase of late sodiumcurrent induces delayed after depolarizations and sustained triggeredactivity in atrial myocytes,” Am. J. Physiol. Heart Circ. Physiol. 2008;294:H2031-H9) in HF lead to leaky ventricular RyR2s (Gonzalez D R, BeigiF, Treuer A V, Hare J M. “Deficient ryanodine receptor S-nitrosylationincreases sarcoplasmic reticulum calcium leak and arrhythmogenesis incardiomyocytes,” Proc. Natl. Acad. Sci. U.S.A. 2007; 104:20612-7; Marx SO, Marks A R. “Dysfunctional ryanodine receptors in the heart: newinsights into complex cardiovascular diseases,” J. Mol. Cell. Cardiol2013; 58:225-31). The oxidation/nitrosylation state of atrial RyR2s inHF has not been fully scrutinized; however, there appear to besignificant differences in atrial versus ventricular E-C coupling, ashas previously been suggested by others (Bootman M D, Smyrnias I, ThulR, Coombes S, Roderick H L. “Atrial cardiomyocyte calcium signaling,”Biochim. Biophys. Acta 2011; 1813:922-34). The majority of the studiesmentioned above have been performed in isolated myocytes; the specificcontribution oxidized I_(Na), RyR2 to the electrophysiologicalcharacteristics of the intact atrium—and how this contributes toarrhythmogenesis—is not known.

Excessively activated CaMKII is implicated in the genesis of HF andarrhythmias. Recent evidence suggests that both CaMKII and 11202increase RyR2 (Marx S O, Marks A R. “Dysfunctional ryanodine receptorsin the heart: new insights into complex cardiovascular diseases,” J.Mol. Cell. Cardiol. 2013; 58:225-31; Niggli E, Ullrich N D, Gutierrez D,Kyrychenko S, Polakova E, Shirokova N. “Posttranslational modificationsof cardiac ryanodine receptors: Ca(2+) signaling and EC-coupling,”Biochim. Biophys. Acta 2013; 1833:866-75) P_(O) (Undrovinas N, MaltsevV, Belardinelli L, Sabbah H, Undrovinas A. “Late sodium currentcontributes to diastolic cell Ca&lt;sup&gt;2+&lt;/sup&gt; accumulationin chronic heart failure,” J. Physiol. Sci. 2010; 60:245-57; Gonzalez DR, Beigi F, Treuer A V, Hare J M. “Deficient ryanodine receptorS-nitrosylation increases sarcoplasmic reticulum calcium leak andarrhythmogenesis in cardiomyocytes,” Proc. Natl. Acad Sci. U.S.A. 2007;104:20612-7; Donoso P, Sanchez G, Bull R, Hidalgo C. “Modulation ofcardiac ryanodine receptor activity by ROS and RNS,” Front. Biosci.(Landmark Ed) 2011; 16:553-67;Terentyev D, Gyorke I, Belevych A E, etal. “Redox modification of ryanodine receptors contributes tosarcoplasmic reticulum Ca²⁺ leak in chronic heart failure,” Circ. Res.2008; 103:1466-72), thereby promoting SR Ca²⁺ leak and arrhythmias(Belevych A E, Terentyev D, Terentyeva R et al “Shortened Ca²⁺ signalingrefractoriness underlies cellular arrhythmogenesis in a postinfarctionmodel of sudden cardiac death,” Circ. Res. 2012; 110:569-77). SinceOS-mediated oxidation of Met 281/282 residues in the regulatory domainof CaMKII transforms CaMKII into a constitutively active form(ox-CaMKII), leading to aberrant phosphorylation of multiple E-Ccoupling proteins (Swaminathan P D, Purohit A, Hund T J, Anderson M E.“Calmodulin-dependent protein kinase linking heart failure andarrhythmias,” Circ. Res. 2012; 110:1661-77; Erickson J R, He B J,Grumbach I M, Anderson M E. “CaMKII in the cardiovascular system:sensing redox states,” Physiol. Rev. 2011; 91:889-915), OS may affectRyR₂ P_(O) both directly (via ROS) and indirectly (via ox-CaMKII)Indeed, expression of ox-CaMKII has been found to be increased in atriaof AF patients, indicating a potential role of ROS induced CaMKIIactivation in AF (Purohit A, Rokita A G, Guan X et at. “OxidizedCa2+/Calmodulin-Dependent Protein Kinase II Triggers AtrialFibrillation,” Circulation 2013; 128:1748-57). CaMKII has also beenshown to modulate the gating of Na_(V)1.5, at least in part byphosphorylation of Na_(V)1.5 at multiple sites (Ashpole N M, Herren A W,Ginsburg K S et al “Ca2+/calmodulin-dependent protein kinase II (CaMKII)regulates cardiac sodium channel NaV1.5 gating by multiplephosphorylation sites,” J. Biol. Chem. 2012; 287:19856-69). A recentstudy demonstrates a fundamental requirement for targeting of CaMKII toa controlling phosphorylation site, S571, on Na_(V)1.5 (Hund T J, KovalO M, Li Jet at. “A beta(IV)-spectrin/CaMKII signaling complex isessential for membrane excitability in mice,” J. Clin. Invest. 2010;120:3508-19).

CaMKII phosphorylation of Na_(V)1.5 is thought to decrease transientI_(Na), but increase I_(Na),late, again thereby contributing to thegenesis of triggered activity (Hashambhoy Y L, Winslow R L, Greenstein JL. “CaMKII-dependent activation of late I_(Na) contributes to cellulararrhythmia in a model of the cardiac myocyte,” Conf Proc. IEEE Eng MedBiol. Soc. 2011; 2011:4665-8). Modeling studies also suggest thatox-CaMKII may create substrate for reentry by regulating conductioncharacteristics of the myocardium (Hashambhoy et al (2011) supra;Christensen M D, Dun W, Boyden P A, Anderson M E, Mohler P J, Hund T J.“Oxidized calmodulin kinase II regulates conduction following myocardialinfarction: a computational analysis,” PLoS Comput. Biol. 2009;5:e1000583), with this substrate thought to be at least partiallymediated by modulation of I_(Na).

A summary of the various mechanisms for reactive oxygen speciesproduction, oxidative stress generation, and development of fibrosis andatrial fibrillation is illustrated in FIG. 1.

SUMMARY

In a first respect, a method of inhibiting oxidative stress in a subjecthaving atrial or ventricular arrhythmias, ventricular failure or heartfailure is provided. The method includes the step of administering aneffective amount of a NOX2 inhibitor agent to the subject, wherein saidadministering is under conditions such that a level of oxidative stressin myocardial tissue is reduced or eliminated.

In a second respect, a method of treating a subject having atrial orventricular arrhythmias, ventricular failure or heart failure isprovided. The method includes the step of administering an effectiveamount of a NOX2 inhibitor agent to the subject, wherein saidadministering is under conditions such that a level of atrial orventricular arrhythmias is reduced or eliminated.

In a third respect, a pharmaceutical composition for inhibitingoxidative stress in a subject having atrial or ventricular arrhythmias,ventricular failure or heart failure is provided. The pharmaceuticalcomposition includes an isolated nucleic acid encoding a small hairpinRNA against NOX2 mRNA (NOX2 rhRNA).

These and other features, objects and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a scheme showing various mechanisms for reactive oxygenspecies production, oxidative stress generation, and development offibrosis and atrial fibrillation.

FIG. 2A depicts O₂ ⁻ production, as measured by lucigenin enhancedchemiluminescence assay, for left atrial appendage (LAA; light bluebars) and posterior left atrium (PLA; dark blue bars) for normal and HFanimals. Statistics key: *, p<0.05; **, p<0.001.

FIG. 2B depicts O₂ ⁻ production (as measured by lucigenin enhancedchemiluminescence assay) in PLA for NADPH oxidase (blue bars),Mitochondrial ROS (red bars), NOS (green bars) and Xanthine oxidase(purple bars) from control and HF animals.

FIG. 3A shows, using western blotting with an anti-gp91 antibody thatNOX2 (91-kDa protein subunit) was increased in the HF PLA relativecontrol PLA (GAPDH control protein (38-kDa) is shown for comparison tonormalize for loading differences in the gel lanes).

FIG. 3B depicts quantitative results of anti-gp91 antibody (NOX2) dataof FIG. 3A.

FIG. 4 shows an exemplary expression vector for expression a NOX2 shRNAunder the control of a U6 PolIII promoter.

FIG. 5 shows that the duration of AF (seconds) is decreased in plasmidencoding NOX2 shRNA-treated animals (“NOX2 shRNA”) compared withlacZ-transfected control animals (“HF (LacZ Control)”).

FIG. 6a shows increased expression of ox-CAMKII in PLA of control HFdogs (green fluorescence represents ox-CAMKII).

FIG. 6B shows decreased expression of ox-CAMKII in the PLA of HF dogstransfected with NOX2 shRNA (green fluorescence represents ox-CAMKII).

FIG. 7 depicts changes in ERP in plasmid encoding NOX2 shRNA-treatedanimals (NOX2 shRNA) compared with lacZ-transfected control animals (HF)for baseline (black bars) and post-pacing (grey bars) conditionedanimals.

FIG. 8A depicts conduction inhomogeneity index (3.25) for PLA fromcontrol HF animals.

FIG. 8B depicts conduction inhomogeneity index (1.5) for PLA fromanimals transfected with plasmid encoding NOX2 rhRNA in PLA tissue.

FIG. 8C depicts statistical differences in conduction inhomogeneityindices for PLA from HF animals at day 1 (black bars) and termination(grey bars).

FIG. 8D depicts no statistical difference in conduction inhomogeneityindices for PLA from animals transfected with plasmid encoding NOX2rhRNA in PLA tissue.

FIG. 9A depicts rtPCR analysis of NOX2 mRNA in PLA from control HFanimals as compared to animals with plasmid encoding NOX2shRNA-transfected PLA.

FIG. 9B depicts representative NOX2 protein levels in PLA from controlHF animals as compared to animals with plasmid encoding NOX2shRNA-transfected PLA (cadherin used as a control protein).

FIG. 10 depicts representative O₂ ⁻ levels attributed to NADPH oxidase(NOX2) in PLA from control HF animals as compared to animals withplasmid encoding NOX2 shRNA-transfected PLA.

FIG. 11A depicts the extent of fibrosis in PLA from control HF animals.

FIG. 11B depicts the extent of fibrosis in PLA from animals with plasmidencoding NOX2 shRNA-transfected PLA.

FIG. 11C depicts a quantitative analysis of the percentage of fibrosisin PLA from control HF animals transfected with control plasmid (thatis, not encoding NOX2 shRNA).

FIG. 12 depicts the onset of AF in a rapid atrial pacing canine modelwhere three control animals experienced sustained AF (blue line)compared with the absence of AF onset in two animals with plasmidencoding NOX2 shRNA-transfected PLA (red line).

DETAILED DESCRIPTION

The present disclosure provides details of the discovery ofpharmaceutical compositions and methods for inhibiting oxidative stressin a subject suffering from atrial fibrillation. The method involvestargeting reducing reactive oxygen species (ROS) generated throughinhibiting the action of NADPH oxidase. The principle is demonstratedusing an NOX2 inhibitor based upon RNA interference (RNAi) with smallhairpin RNAs directed against the NOX2 mRNA (NOX2 shRNA). Pharmaceuticalcompositions based upon NOX2 shRNA is shown in the method to inhibitexpression of the NOX2 gene, thereby leading to decreased production ofNADPH oxidase in cardiac cells containing the injected NOX2 shRNAs.Referring to FIG. 1, lower NADPH oxidase production can lead to lowerlevels of ROS produced, lower extents of oxidative stress, decreasedextents of atrial fibrosis and lower atrial fibrillation (AF). However,one skilled in the art will appreciate that the principle can be readilyextended to other NOX2 inhibitors with undue experimentation and have areasonable expectation of achieving similar results as described herein.Details of the pharmaceutical compositions and methods are presented ingreater detail in this disclosure.

TERMINOLOGY AND DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. With respect tothe use of substantially, any plural and/or singular terms herein, thosehaving skill in the art can translate from the plural as is appropriateto the context and/or application. The various singular/pluralpermutations may be expressly set forth herein for the sake of clarity.

Terms used herein are intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.).

The phrase “such as” should be interpreted as “for example, including.”

Furthermore, in those instances where a convention analogous to “atleast one of A, B and C, etc.” is used, in general such a constructionis intended in the sense of one having ordinary skill in the art wouldunderstand the convention (e.g., “a system having at least one of A, Band C” would include but not be limited to systems that have A alone, Balone, C alone, A and B together, A and C together, B and C together,and/or A, B, and C together.). It will be further understood by thosewithin the art that virtually any disjunctive word and/or phrasepresenting two or more alternative terms, whether in the description orfigures, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or ‘B or “A and B.”

All language such as “up to,” “at least,” “greater than,” “less than,”and the like, include the number recited and refer to ranges which cansubsequently be broken down into subranges as discussed above.

A range includes each individual member. Thus, for example, a grouphaving 1-3 members refers to groups having 1, 2, or 3 members.Similarly, a group having 6 members refers to groups having 1, 2, 3, 4,or 6 members, and so forth.

The phrase “small hairpin RNA” and the term “shRNA”, as used herein,refer to a unimolecular RNA-containing polynucleotide that is capable ofperforming RNAi and that includes a sense sequence, a loop, and anantisense sequence. The sense and antisense sequences are sometimesreferred to herein as the first region and second region. As describedherein, the sense and antisense sequences can be in differentorientations with respect to one another in an shRNA of the invention(an L or R shRNA). Thus, if the first region of an shRNA is the sensesequence then the second region is the antisense region, and vice versa.Preferably, the sense and antisense sequences are substantiallycomplementary to each other (about 80% complementary). The antisensesequence can be about 16 to about 22 nucleotides in length, e.g., about16 to 19 nucleotides, and more preferably 18 to 19 nucleotides inlength. The sense sequence can be about 11 to about 22 nucleotides inlength, and more preferably 17 to 19 nucleotides in length. An shRNA(and other RNAi agents) are “specific” for a target gene when theantisense sequence (of about 16 to 22 nucleotides is substantiallycomplementary to the target gene (or target RNA, e.g., target mRNA). Bysubstantially complementary is meant that the antisense sequence is atleast 80% complementary to the target gene (or gene product). Thus, insome embodiments, the antisense sequence that is complementary to thetarget gene can contain mismatches to the target. The sequence can bevaried to target one or more genetic variants or phenotypes of a target,e.g., a viral target, by altering the targeting sequence to becomplementary to the sequence of the genetic variant or phenotype. AnshRNA may have a loop as long as, for example, 0 to about 24 nucleotidesin length, preferably 0 to about 10 nucleotides in length, 0 to 6nucleotides in length, e.g., 2 nucleotides in length. The sequence ofthe loop can include nucleotide residues unrelated to the target. In oneparticularly preferred embodiment, the loop is 5′-UU-3′. In someembodiments it may include non-nucleotide moieties. In yet otherembodiments, the loop does not include any non-nucleotides moieties.Optionally, the shRNA can have an overhang region of 2 bases on 3′ endof the molecule. The shRNA can also comprise RNAs with stem-loopstructures that contain mismatches and/or bulges. The sense sequencethat is homologous to the target can differ at about 0 to about 5 sitesby having mismatches, insertions, or deletions of from about 1 to about5 nucleotides, as is the case, for example, with naturally occurringmicroRNAs. RNAs that comprise any of the above structures can includestructures where the loops comprise nucleotides, non-nucleotides, orcombinations of nucleotides and non-nucleotides.

Additionally, the phrase “small hairpin RNA” and the term “shRNA”include nucleic acids that also contain moieties other thanribonucleotide moieties, including, but not limited to, modifiednucleotides, modified internucleotide linkages, non-nucleotides,deoxynucleotides and analogs of the nucleotides mentioned thereof.

Additionally, the term “L shRNA” refers to an shRNA comprising a sensesequence that is connected through a loop to the 3′ end of theantisense.

Additionally, the term “R shRNA” refers to an shRNA molecule comprisingan antisense sequence that is connected through a loop to the 3′ end ofthe sense sequence.

The phrase “antisense sequence”, as used herein, refers to apolynucleotide or region of a polynucleotide that is substantiallycomplementary (e.g., 80% or more) or 100% complementary to a targetnucleic acid of interest. An antisense sequence can be composed of apolynucleotide region that is RNA, DNA or chimeric RNA/DNA. Anynucleotide within an antisense sequence can be modified by includingsubstituents coupled thereto, such as in a 2′ modification. Theantisense sequence can also be modified with a diverse group of smallmolecules and/or conjugates. For example, an antisense sequence may becomplementary, in whole or in part, to a molecule of messenger RNA, anRNA sequence that is not mRNA (e.g., tRNA, rRNA, hnRNA, negative andpositive stranded viral RNA and its complementary RNA) or a sequence ofDNA that is either coding or non-coding.

The phrase “sense sequence”, as used herein, refers to a polynucleotideor region that has the same nucleotide sequence, in whole or in part, asa target nucleic acid such as a messenger RNA or a sequence of DNA. Whena sequence is provided, by convention, unless otherwise indicated, it isthe sense sequence (or region), and the presence of the complementaryantisense sequence (or region) is implicit.

The term “complementary”, as used herein, refers to the ability ofpolynucleotides to form base pairs with one another. Base pairs aretypically formed by hydrogen bonds between nucleotide units inantiparallel polynucleotide strands or regions. Complementarypolynucleotide strands or regions can base pair in the Watson-Crickmanner (e.g., A to T, A to U, C to G), or in any other manner thatallows for the formation of stable duplexes.

“Perfect complementarity” or “100% complementarity”, as used herein,refers to the situation in which each nucleotide unit of onepolynucleotide strand or region can hydrogen bond with each nucleotideunit of a second polynucleotide strand or region. Less than perfectcomplementarity refers to the situation in which some, but not all,nucleotide units of two strands or two regions can hydrogen bond witheach other. For example, for two 19-mers, if 17 base pairs on eachstrand or each region can hydrogen bond with each other, thepolynucleotide strands exhibit 89.5% complementarity. Substantialcomplementarity refers to polynucleotide strands or regions exhibitingabout 80% or greater complementarity.

The term “deoxynucleotide”, as used herein, refers to a nucleotide orpolynucleotide lacking an OH group at the 2′ or 3′ position of a sugarmoiety with appropriate bonding and/or 2′, 3′ terminal dideoxy, insteadhaving a hydrogen bonded to the 2′ and/or 3′ carbon.

The terms “deoxyribonucleotide” and “DNA”, as used herein, refer to anucleotide or polynucleotide comprising at least one ribosyl moiety thathas an H at its 2′ position of a ribosyl moiety instead of an OH.

In some embodiments, an shRNA described herein optionally includes atleast one conjugate moiety.

The term “alkyl”, as used herein, refers to a hydrocarbyl moiety thatcan be saturated or unsaturated, and substituted or unsubstituted. Itmay comprise moieties that are linear, branched, cyclic and/orheterocyclic, and contain functional groups such as ethers, ketones,aldehydes, carboxylates, etc. Unless otherwise specified, alkyl groupsare not cyclic, heterocyclic, or comprise functional groups.

Exemplary alkyl groups include, but are not limited to, substituted andunsubstituted groups of methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicoyl andalkyl groups of higher number of carbons, as well as 2-methylpropyl,2-methyl-4-ethylbutyl, 2,4-diethylpropyl, 3-propylbutyl,2,8-dibutyldecyl, 6,6-dimethyloctyl, 6-propyl-6-butyloctyl,2-methylbutyl, 2-methylpentyl, 3-methylpentyl, and 2-ethylhexyl. Theterm alkyl also encompasses alkenyl groups, such as vinyl, allyl,aralkyl and alkynyl groups. Unless otherwise specified, alkyl groups arenot substituted.

Substitutions within an alkyl group, when specified as present, caninclude any atom or group that can be tolerated in the alkyl moiety,including but not limited to halogens, sulfurs, thiols, thioethers,thioesters, amines (primary, secondary, or tertiary), amides, ethers,esters, alcohols and oxygen. The alkyl groups can by way of example alsocomprise modifications such as azo groups, keto groups, aldehyde groups,carboxyl groups, nitro, nitroso or nitrile groups, heterocycles such asimidazole, hydrazine or hydroxylamino groups, isocyanate or cyanategroups, and sulfur containing groups such as sulfoxide, sulfone,sulfide, and disulfide. Unless otherwise specified, alkyl groups do notcomprise halogens, sulfurs, thiols, thioethers, thioesters, amines,amides, ethers, esters, alcohols, oxygen, or the modifications listedabove.

Further, alkyl groups may also contain hetero substitutions, which aresubstitutions of carbon atoms, by for example, nitrogen, oxygen orsulfur. Heterocyclic substitutions refer to alkyl rings having one ormore heteroatoms. Examples of heterocyclic moieties include but are notlimited to morpholino, imidazole, and pyrrolidino. Unless otherwisespecified, alkyl groups do not contain hetero substitutions or alkylrings with one or more heteroatoms (i.e., heterocyclic substitutions).

The preferred alkyl group for a 2′ modification is a methyl group withan O-linkage to the 2′ carbon of a ribosyl moiety, i.e., a 2′ O-alkylthat comprises a 2′-O-methyl group.

The phrase “2′-O-alkyl modified nucleotide”, as used herein, refers to anucleotide unit having a sugar moiety, for example a deoxyribosyl moietythat is modified at the 2′ position such that an oxygen atom is attachedboth to the carbon atom located at the 2′ position of the sugar and toan alkyl group. In various embodiments, the alkyl moiety consistsessentially of carbons and hydrogens. A particularly preferredembodiment is one wherein the alkyl moiety is methyl.

As used herein, the term “2′ carbon modification” refers to a nucleotideunit having a sugar moiety, for example a moiety that is modified at the2′ position of the sugar subunit. A “2′-O-alkyl modified nucleotide” ismodified at this position such that an oxygen atom is attached both tothe carbon atom located at the 2′ position of the sugar and to an alkylgroup, e.g., 2′-O-methyl, 2′O-ethyl, 2′-O-propyl, 2′-O-isopropyl,2′-O-butyl, 2′-O-isobutyl, 2′-O-ethyl-O-methyl(—OCH₂CH₂OCH₃), and2′-o-ethyl-OH(—OCH₂CH₂OH). A “2′ carbon sense sequence modification”, asused herein, refers to a modification at the 2′ carbon position of anucleotide on the sense sequence. A “2′ carbon antisense sequencemodification”, as used herein, refers to a modification at the 2′ carbonposition of a nucleotide on the antisense sequence.

The term “nucleotide”, as used herein, refers to a ribonucleotide or adeoxyribonucleotide or modified from thereof, as well as an analogthereof. Nucleotides include species that comprise purines, e.g.,adenine, hypoxanthine, guanine, and their derivatives and analogs, aswell as pyrimidines, e.g., cytosine, uracil, thymine, and theirderivatives and analogs. Preferably, a “nucleotide” comprises acytosine, uracil, thymine, adenine, or guanine moiety.

Nucleotide analogs include nucleotides having modifications in thechemical structure of the base, sugar and/or phosphate, including, butnot limited to, 5-position pyrimidine modifications, 8-position purinemodifications, modifications at cytosine exocyclic amines, andsubstitution of 5-bromo-uracil; and 2′-position sugar modifications,including but not limited to, sugar-modified ribonucleotides in whichthe 2′-OH is replaced by a group such as an H, OR, R, halo, SH, SR,NH.sub.2, NHR, NR.sub.2, or CN, wherein R is an alkyl moiety as definedherein. Nucleotide analogs also include nucleotides with bases such asinosine, queuosine, xanthine, sugars such as 2′-methyl ribose,non-natural phosphodiester linkages such as methylphosphonates,phosphorothioates and peptides.

Modified bases refer to nucleotide bases such as, for example, adenine,guanine, cytosine, thymine, and uracil, xanthine, inosine, and queuosinethat have been modified by the replacement or addition of one or moreatoms or groups. Some examples of types of modifications includenucleotides that are modified with respect to the base moieties, includebut are not limited to, alkylated, halogenated, thiolated, aminated,amidated, or acetylated bases, in various combinations. More specificmodified bases include, for example, 5-propynyluridine,5-propynylcytidine, 6-methyladenine, 6-methylguanine,N,N,-dimethyladenine, 2-propyladenine, 2-propylguanine, 2′-aminoadenine,1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine andother nucleotides having a modification at the 5 position,5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine,4-acetylcytidine, 1-methyladenosine, 2-methyladenosien,3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine,2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine,deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine,6-azothymidine, 5-methyl-2-thiouridine, other thio bases such as2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine,pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthylgroups, any 0- and N-alkylated purines and pyrimidines such asN6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyaceticacid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groupssuch as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines thatact as G-clamp nucleotides, 8-substituted adenines and guanines,5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkylnucleotides, carboxyalkylaminoalkyl nucleotides, andalkylcarbonylalkylated nucleotides. Modified nucleotides also includethose nucleotides that are modified with respect to the sugar moiety, aswell as nucleotides having sugars or analogs thereof that are notribosyl. For example, the sugar moieties may be, or be based on,mannoses, arabinoses, glucopyranoses, galactopyranoses, 4-thioribose,and other sugars, heterocycles, or carbocycles. The term nucleotideanalog also includes what are known in the art as universal bases. Byway of example, universal bases include but are not limited to3-nitropyrrole, 5-nitroindole, or nebularine.

Further, the term nucleotide analog also includes those species thathave a detectable label, such as, for example, a radioactive orfluorescent moiety, or mass label attached to the nucleotide.

The term “overhang”, as used herein, refers to terminal non-base pairingnucleotide(s) resulting from one strand or region extending beyond theterminus of the complementary strand to which the first strand or regionforms a duplex. One or more polynucleotides that are capable of forminga duplex through hydrogen bonding can have overhangs. Thesingle-stranded region extending beyond the 3′ end of the duplex isreferred to as an overhang.

The term “ribonucleotide” and the phrase “ribonucleic acid” (RNA), asused herein, refer to a modified or unmodified nucleotide orpolynucleotide comprising at least one ribonucleotide unit. Aribonucleotide unit comprises an oxygen attached to the 2′ position of aribosyl moiety having a nitrogenous base attached in N-glycosidiclinkage at the 1′ position of a ribosyl moiety, and a moiety that eitherallows for linkage to another nucleotide or precludes linkage.

The phrase “heating and snap cooling”, as used herein, refers to atwo-step procedure that involves heat-denaturing nucleic acids in asample followed by rapid cooling. For example, tubes that contain shRNAsolutions are denatured in a 95.degree. C. heat block for 4 to 5 minutesfollowed by immediately placing the tubes into an ice-water bath for 30minutes. Such “heating and snap cooling” favors the formation of shRNAmonomers over multimers.

As used herein, the term “pharmaceutically acceptable” is a carrier,diluent, excipient, and/or salt that is compatible with the otheringredients of the formulation, and not deleterious to the recipientthereof The active ingredient for administration may be present as apowder or as granules; as a solution, a suspension or an emulsion or asdescribed elsewhere throughout the specification.

The phrase “pharmaceutically acceptable carrier”, as used herein, meansa pharmaceutically acceptable salt, solvent, suspending agent or vehiclefor delivering a composition of the present disclosure to the animal orhuman. The carrier may be liquid, semisolid or solid, and is oftensynonymously used with diluent, excipient or salt. The phrase“pharmaceutically acceptable” means that an ingredient, excipient,carrier, diluent or component disclosed is one that is suitable for usewith humans and/or animals without undue adverse side effects (such astoxicity, irritation, and allergic response) commensurate with areasonable benefit/risk ratio. See Remington's Pharmaceutical Sciences16.sup.th edition, Osol, A. Ed (1980) (incorporated herein by referencein its entirety).

As used herein, the term “operably-linked” refers to the association ofnucleic acid sequences on a polynucleotide so that the function of oneof the sequences is affected by another. For example, a regulatory DNAsequence is said to be “operably linked to” a DNA sequence that codesfor an RNA (“an RNA coding sequence” or “shRNA encoding sequence”) or apolypeptide if the two sequences are situated such that the regulatoryDNA sequence affects expression of the coding DNA sequence (i.e., thatthe coding sequence or functional RNA is under the transcriptionalcontrol of the promoter). Coding sequences can be operably-linked toregulatory sequences in sense or antisense orientation. An RNA codingsequence refers to a nucleic acid that can serve as a template forsynthesis of an RNA molecule such as an shRNA. Preferably, the RNAcoding region is a DNA sequence.

As used herein, the term “promoter” refers to a nucleotide sequence,usually upstream (5′) to its coding sequence, which directs and/orcontrols the expression of the coding sequence by providing therecognition for RNA polymerase and other factors required for propertranscription. “Promoter” includes a minimal promoter that is a shortDNA sequence comprised of a TATA-box and other sequences that serve tospecify the site of transcription initiation, to which regulatoryelements are added for control of expression. “Promoter” also refers toa nucleotide sequence that includes a minimal promoter plus regulatoryelements that is capable of controlling the expression of a codingsequence or functional RNA. This type of promoter sequence consists ofproximal and more distal upstream elements, the latter elements oftenreferred to as enhancers. Accordingly, an “enhancer” is a DNA sequencethat stimulates promoter activity and may be an innate element of thepromoter or a heterologous element inserted to enhance the level ortissue specificity of a promoter. It is capable of operating in bothorientations (sense or antisense), and is capable of functioning evenwhen moved either upstream or downstream from the promoter. Bothenhancers and other upstream promoter elements bind sequence-specificDNA-binding proteins that mediate their effects. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature, oreven be comprised of synthetic DNA segments. A promoter may also containDNA sequences that are involved in the binding of protein factors thatcontrol the effectiveness of transcription initiation in response tophysiological or developmental conditions. Any promoter known in the artwhich regulates the expression of the shRNA or RNA coding sequence isenvisioned in the practice of the invention.

As used herein, the term “reporter element” or “marker” is meant apolynucleotide that encodes a polypeptide capable of being detected in ascreening assay. Examples of polypeptides encoded by reporter elementsinclude, but are not limited to, lacZ, GFP, luciferase, andchloramphenicol acetyltransferase. See, for example, U.S. Pat. No.7,416,849. Many reporter elements and marker genes are known in the artand envisioned for use in the inventions disclosed herein.

As used herein, the term “RNA transcript” refers to the productresulting from RNA polymerase catalyzed transcription of a DNA sequence.“Messenger RNA transcript (mRNA)” refers to the RNA that is withoutintrons and that can be translated into protein by the cell.

As used herein, the term “shRNA” (small hairpin RNA) refers to an RNAduplex wherein a portion of the RNA is part of a hairpin structure(shRNA). In addition to the duplex portion, the hairpin structure maycontain a loop portion positioned between the two sequences that formthe duplex. The loop can vary in length. In some embodiments the loop is5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpinstructure can also contain 3′ or 5′ overhang portions. In some aspects,the overhang is a 3′ or a 5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides inlength. In one aspect of this invention, a nucleotide sequence in thevector serves as a template for the expression of a small hairpin RNA,comprising a sense region, a loop region and an antisense region.Following expression the sense and antisense regions form a duplex. Itis this duplex, forming the shRNA, which hybridizes to, for example, theNOX2 mRNA and reduces expression of NADPH oxidase, lowering ROS andoxidative stress levels, fibrosis and AF.

As used herein, the term “knock-down” or “knock-down technology” refersto a technique of gene silencing in which the expression of a targetgene or gene of interest is reduced as compared to the gene expressionprior to the introduction of the siRNA, which can lead to the inhibitionof production of the target gene product. “Double knockdown” is theknockdown of two genes. The term “reduced” is used herein to indicatethat the target gene expression is lowered by 0.1-100%. For example, theexpression may be reduced 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, or even 99%. The expression may bereduced by any amount (%) within those intervals, such as for example,2-4, 11-14, 16-19, 21-24, 26-29, 31-34, 36-39, 41-44, 46-49, 51-54,56-59, 61-64, 66-69, 71-74, 76-79, 81-84, 86-89, 91-94, 96, 97, 98 or99. Knock-down of gene expression can be directed by the use of shRNAs.

As used herein, the term “treating” refers to ameliorating at least onesymptom of, curing and/or preventing the development of a disease ordisorder such as for example, but not limited to, cardiac fibrosis andAF.

As used herein, the term “vector” refers to any viral or non-viralvector, as well as any plasmid, cosmid, phage or binary vector in doubleor single stranded linear or circular form that may or may not be selftransmissible or mobilizable, and that can transform prokaryotic oreukaryotic host cells either by integration into the cellular genome orwhich can exist extrachromosomally (e.g., autonomous replicating plasmidwith an origin of replication). Any vector known in the art isenvisioned for use in the practice of this invention.

The modal verb “may” refers to the preferred use or selection of one ormore options or choices among the several described embodiments orfeatures contained within the same. Where no options or choices aredisclosed regarding a particular embodiment or feature contained in thesame, the modal verb “may” refers to an affirmative act regarding how tomake or use and aspect of a described embodiment or feature contained inthe same, or a definitive decision to use a specific skill regarding adescribed embodiment or feature contained in the same. In this lattercontext, the modal verb “may” has the same meaning and connotation asthe auxiliary verb “can.”

Compositions for Inhibiting NOX2 Gene Expression in Posterior LeftAtrium (PLA) Tissue

In a one aspect, a pharmaceutical composition for treating oxidativestress in atrial fibrillation is provided. The pharmaceuticalcomposition includes a small hairpin RNA (shRNA) directed against a NOX2gene (“NOX2 shRNA”). The shRNA can be a unimolecular RNA that includes asense sequence, a loop region, and an antisense sequence (sometimesreferred to as first and second regions, as noted above), which togetherform a hairpin loop structure. Preferably, the antisense and sensesequences are substantially complementary to one other (about 80%complementary or more), where in certain embodiments the antisense andsense sequences are 100% complementary to each other. In certainembodiments, the antisense and sense sequences are too short to beprocessed by Dicer, and hence act through an alternative pathway to thatof longer double-stranded RNAs (e.g., shRNAs having antisense and sensesequences of about 16 to about 22 nucleotides in length, e.g., between18 and 19 nucleotides in length (e.g., an sshRNA). Additionally, theantisense and sense sequences within a unimolecular RNA of the inventioncan be the same length, or differ in length by less than about 9 bases.The loop can be any length, with the preferred length being from 0 to 4nucleotides in length or an equivalent length of non-nucleotidic linker,and more preferably 2 nucleotides or an equivalent length ofnon-nucleotidic linker (e.g., a non-nucleotide loop having a lengthequivalent to 2 nucleotides). In one embodiment, the loop is: 5′-UU-3′(rUrU) or 5′-tt-3′, where “t” represents deoxythymidine (dTdT). Withinany shRNA hairpin, a plurality of the nucleotides are ribonucleotides.In the case of a loop of zero nucleotides, the antisense sequence islinked directly to the sense sequence, with part of one or both strandsforming the loop. In a preferred embodiment of a zero-nt loop shRNA, theantisense sequence is about 18 or 19 nt and the sense sequence isshorter than the antisense sequence, so that one end of the antisensesequence forms the loop.

A hairpin of representative shRNA's can be organized in either aleft-handed (L) hairpin (i.e., 5′-antisense-loop-sense-3′) or aright-handed (R) hairpin (i.e., 5′-sense-loop-antisense-3′).Furthermore, an shRNA may also contain overhangs at either the 5′ or 3′end of either the sense sequence or the antisense sequence, dependingupon the organization of the hairpin. Preferably, if there are anyoverhangs, they are on the 3′ end of the hairpin and comprise between 1to 6 bases. The presence of an overhang is preferred for R-typehairpins, in which case a 2-nt overhang is preferred, and a UU or ttoverhang is most preferred.

Modifications can be added to enhance shRNA stability, functionality,and/or specificity and to minimize immunostimulatory properties. Forexample, the overhangs can be unmodified, or can contain one or morespecificity or stabilizing modifications, such as a halogen or O-alkylmodification of the 2′ position, or internudeotide modifications such asphosphorothioate modification. The overhangs can be ribonucleic acid,deoxyribonucleic acid, or a combination of ribonucleic acid anddeoxyribonucleic acid.

In another non-limiting example of modifications that can be applied toleft handed hairpins, 2′-O-methyl modifications (or other 2′modifications, including but not limited to other 2′-O-alkylmodifications) can be added to nucleotides at position 15, 17, or 19from the 5′ antisense terminus of the hairpin, or any two of thosepositions, or all three, as well as to the loop nucleotides and to everyother nucleotide of the sense sequence except for nucleotides 9, 10 and11 from the 5′-most nucleotide of the sense sequence (also called the9.sup.th, 10.sup.th, and 11.sup.th nucleotides), which should have nomodifications that block “slicing” activity. Any single modification orgroup of modifications described in the preceding sentence can be usedalone or in combination with any other modification or group ofmodifications cited.

Ui-Tei, K. et al. (Nucl. Acids Res. (2008) 36 (22): 7100-7109) observedthat the specificity of siRNAs can be increased by modifying the seedregion of one or both strands. Such modifications are applicable toshRNA's of the present disclosure. In another non-limiting example ofmodifications that can be applied to hairpins, nt 1-6 of the antisensesequence and nt 14-19 of the sense sequence can be 2′-O-methylated toreduce off-target effects. In a preferred embodiment, only nt 1-6 aremodified from 2′-OH to 2′-H or 2′-O-alky.

As the sense sequence of an shRNA can potentially enter RISC and competewith the antisense (targeting) strand, modifications that prevent sensesequence phosphorylation are valuable in minimizing off-targetsignatures. Thus, desirable chemical modifications that preventphosphorylation of the 5′ carbon of the 5′-most nucleotide ofright-handed shRNA of the invention can include, but are not limited to,modifications that: (1) add a blocking group (e.g., a 5′-O-alkyl) to the5′ carbon; or (2) remove the 5′-hydroxyl group (e.g., 5′-deoxynucleotides) (see, e.g., WO 2005/078094).

In addition to modifications that enhance specificity, modificationsthat enhance stability can also be added. In one embodiment,modifications comprising 2′-O-alkyl groups (or other 2′ modifications)can be added to one or more, and preferably all, pyrimidines (e.g., Cand/or U nucleotides) of the sense sequence. Modifications such as 2′ For 2′-O-alkyl of some or all of the Cs and Us of the sensesequence/region, respectively, or the loop structure, can enhance thestability of the shRNA molecules without appreciably altering targetspecific silencing. It should be noted that while these modificationsenhance stability, it may be desirable to avoid the addition of thesemodification patterns to key positions in the hairpin in order to avoiddisruption of RNAi (e.g., that interfere with “slicing” activity).

Additional stabilization modifications to the phosphate backbone may beincluded in the shRNAs in some embodiments of the present invention. Forexample, at least one phosphorothioate, phosphordithioate, and/ormethylphosphonate may be substituted for the phosphate group at some orall 3′ positions of nucleotides in the shRNA backbone, or any particularsubset of nucleotides (e.g., any or all pyrimidines in the sensesequence of the oligonucleotide backbone), as well as in any overhangs,and/or loop structures present. These modifications may be usedindependently or in combination with the other modifications disclosedherein.

Description of modified shRNAs of interest can be found in the followingreferences, both of which are incorporated herein by reference in theirentirety: Q. Ge, H. Ilves, A. Dallas, P. Kumar, J. Shorenstein, S. A.Kazakov, and B. H. Johnston (2010) Minimal-length short hairpin RNAs:The Relationship of Structure and RNAi Activity. RNA 16(1):106-17 (EpubDec. 1, 2009); and Q. Ge, A. Dallas, H. Ilves, J. Shorenstein, M. A.Behlke, and B. H. Johnston (2010) Effects of Chemical Modification onthe Potency, Serum Stability, and Immunostimulatory Properties of ShortshRNAs. RNA 16(1):118-30 (Epub Nov. 30, 2009).

Modified shRNAs according to aspects of the present invention mayinclude additional chemical modifications for any of a variety ofpurposes, including 3′ cap structures (e.g., an inverteddeoxythymidine), detectable labels conjugated to one or more positionsin the shRNA (e.g., fluorescent labels, mass labels, radioactive labels,etc.), or other conjugates that can enhance delivery, detection,function, specificity, or stability (e.g., amino acids, peptides,proteins, sugars, carbohydrates, lipids, polymers, nucleotides,polynucleotides, etc.). Combinations of additional chemicalmodifications may be employed as desired by the user.

Suitable NOX2 shRNAs include those nucleic acids ranging from about 20nucleotides to about 80 nucleotides in length, wherein a portion of thenucleic acids have a double-stranded structural domain ranging fromabout 15 nucleotides to about 25 nucleotides in length. In some aspects,the shRNA can include modified bases or phosphodiester backbones toimpart stability of the shRNA inside tissues and cells. An exemplaryNOX2 shRNA includes SEQ ID NO:1 presented in Table 1.

TABLE 1 Exemplary NOX2 shRNA (SEQ ID NO: 1)  5′ → 3′ Nucleotide Sequence  TATCCATTTCCAAGTCATAGG

shRNA: Synthesis

As is generally known in the art, commonly used oligonucleotides areoligomers or polymers of ribonucleic acid or deoxyribonucleic acidhaving a combination of naturally-occurring purine and pyrimidine bases,sugars and covalent linkages between nucleosides including a phosphategroup in a phosphodiester linkage. However, it is noted that the term“oligonucleotides” also encompasses various non-naturally occurringmimetics and derivatives, i.e., modified forms, of naturally occurringoligonucleotides, as described herein.

shRNA molecules of the invention can be prepared by any method known inthe art for the synthesis of DNA and RNA molecules. These includetechniques for chemically synthesizing oligodeoxy-ribonucleotides andoligo-ribonucleotides well known in the art such as for example solidphase phosphoramidite chemical synthesis. Alternatively, RNA moleculescan be generated by in vitro and in vivo transcription of DNA sequencesencoding the antisense RNA molecule. Such DNA sequences may beincorporated into a wide variety of vectors that incorporate suitableRNA polymerase promoters such as the T7 or SP6 polymerase promoters.Alternatively, antisense cDNA constructs that synthesize antisense RNAconstitutively or inducibly, depending on the promoter used, can beintroduced stably into cell lines.

shRNA molecules can be chemically synthesized using appropriatelyprotected ribonucleoside phosphoramidites and a conventional DNA/RNAsynthesizer. Custom shRNA synthesis services are available fromcommercial vendors such as Ambion (Austin, Tex., USA) and DharmaconResearch (Lafayette, Colo., USA). See, for example, U.S. Pat. No.7,410,944.

Various well-known modifications to the DNA molecules can be introducedas a means of increasing intracellular stability and half-life. Possiblemodifications include, but are not limited to, the addition of flankingsequences of ribo- or deoxy-nucleotides to the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the oligodeoxyribonucleotide backbone.An antisense nucleic acid of the invention can be constructed usingchemical synthesis or enzymatic ligation reactions using proceduresknown in the art. An antisense oligonucleotide can be chemicallysynthesized using naturally-occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedbetween the antisense and sense nucleic acids (e.g., phosphorothioatederivatives and acridine substituted nucleotides can be used).

The shRNA molecules of the invention can be various modified equivalentsof the structures of any NOX2 shRNA. A “modified equivalent” means amodified form of a particular shRNA molecule having the sametarget-specificity (i.e., recognizing the same mRNA molecules thatcomplement the unmodified particular shRNA molecule). Thus, a modifiedequivalent of an unmodified shRNA molecule can have modifiedribonucleotides, that is, ribonucleotides that contain a modification inthe chemical structure of an unmodified nucleotide base, sugar and/orphosphate (or phosphodiester linkage). See, for example, U.S. Pat. No.7,410,944.

Preferably, modified shRNA molecules contain modified backbones ornon-natural internucleoside linkages, e.g., modifiedphosphorous-containing backbones and non-phosphorous backbones such asmorpholino backbones; siloxane, sulfide, sulfoxide, sulfone, sulfonate,sulfonamide, and sulfamate backbones; formacetyl and thioformacetylbackbones; alkene-containing backbones; methyleneimino andmethylenehydrazino backbones; amide backbones, and the like. See, forexample, U.S. Pat. No. 7,410,944.

Examples of modified phosphorous-containing backbones include, but arenot limited to phosphorothioates, phosphorodithioates, chiralphosphorothioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates, thionoalkylphosphonates, phosphinates, phosphoramidates,thionophosphoramidates, thionoalkylphosphotriesters, andboranophosphates and various salt forms thereof. See, for example, U.S.Pat. No. 7,410,944.

Examples of the non-phosphorous containing backbones described above areknown in the art, e.g., U.S. Pat. No. 5,677,439, each of which is hereinincorporated by reference. See, for example, U.S. Pat. No. 7,410,944.

Modified forms of shRNA compounds can also contain modified nucleosides(nucleoside analogs), i.e., modified purine or pyrimidine bases, e.g.,5-substituted pyrimidines, 6-azapyrimidines, pyridin-4-one,pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine(e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), 2-thiouridine, 4-thiouridine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine,5-methylaminomethyluridine, 5-methylcarbonylmethyl uridine,5-methyloxyuridine, 5-methyl-2-thiouridine, 4-acetylcytidine,3-methylcytidine, propyne, quesosine, wybutosine, wybutoxosine,beta-D-galactosylqueosine, N-2, N-6 and 0-substituted purines, inosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,2-methyladenosine, 2-methylguanosine, N6-methyladenosine,7-methylguanosine, 2-methylthio-N-6-isopentenyl adenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives, and the like. See, for example, U.S. Pat. No.7,410,944.

In addition, modified shRNA compounds can also have substituted ormodified sugar moieties, e.g., 2′-O-methoxyethyl sugar moieties. See,for example, U.S. Pat. No. 7,410,944.

Preferably, the 3′ overhangs of the shRNAs of the present invention aremodified to provide resistance to cellular nucleases. In one embodimentthe 3′ overhangs comprise 2′-deoxyribonucleotides.

Additional shRNA compounds targeted at different sites of the mRNAcorresponding to NOX2. Additionally, to assist in the design of shRNAsfor the efficient RNA interference (RNAi)-mediated silencing of anytarget gene, several shRNA supply companies maintain web-based designtools that utilize these general guidelines for “picking” shRNAs whenpresented with the mRNA or coding DNA sequence of the target gene.Examples of such tools can be found at the web sites of Dharmacon, Inc.(Lafayette, Colo.), Ambion, Inc. (Austin, Tex.). As an example, pickingshRNAs involves choosing a site/sequence unique to the target gene(i.e., sequences that share no significant homology with genes otherthan the one being targeted), so that other genes are not inadvertentlytargeted by the same shRNA designed for this particular target sequence.

Another criterion to be considered is whether or not the target sequenceincludes a known polymorphic site. If so, shRNAs designed to target oneparticular allele may not effectively target another allele, sincesingle base mismatches between the target sequence and its complementarystrand in a given shRNA can greatly reduce the effectiveness ofRNAi-induced by that shRNA. Given that target sequence and such designtools and design criteria, an ordinarily skilled artisan apprised of thepresent disclosure should be able to design and synthesized additionalsihRNA compounds useful in reducing the mRNA level of NOX2.

shRNA: Administration

The present invention provides a composition of a polymer or excipientand one or more vectors encoding one or more shRNA molecules. The vectorcan be formulated into a pharmaceutical composition with suitablecarriers and administered into a mammal using any suitable route ofadministration.

Because of this precision, side effects typically associated withtraditional drugs can be reduced or eliminated. In addition, shRNA arerelatively stable, and like antisense, they can also be modified toachieve improved pharmaceutical characteristics, such as increasedstability, deliverability, and ease of manufacture. Moreover, becauseshRNA molecules take advantage of a natural cellular pathway, i.e., RNAinterference, they are highly efficient in destroying targeted mRNAmolecules. As a result, it is relatively easy to achieve atherapeutically effective concentration of an shRNA compound in asubject. See, for example, U.S. Pat. No. 7,410,944.

shRNA compounds may be administered to mammals by various methodsthrough different routes. They can also be delivered directly to aparticular organ or tissue by any suitable localized administrationmethods such as direct injection into a target tissue. Preferably, shRNAcompounds can be electroporated into cells following their injectiondirectly into the target tissue. Alternatively, they may be deliveredencapsulated in liposomes, by iontophoresis, or by incorporation intoother vehicles such as hydrogels, cyclodextrins, biodegradablenanocapsules, and bioadhesive microspheres.

In vivo inhibition of specific gene expression by RNAi injectedintravenously has been achieved in various organisms including mammals.See, for example, Song E. et al “RNA interference targeting Fas protectsmice from fulminant hepatitis,” Nature Medicine, 9:347-351 (2003). Oneroute of administration of shRNA molecules of the invention includesdirect injection of the vector at a desired tissue site, such as forexample, into diseased or non-diseased cardiac tissue, into fibroticheart tissue, such as fibrotic PLA tissue. Generally, however, NOX2shRNAs or expression vectors encoding NOX2 shRNAs can be directlyinjected into any atrial tissue to effectively knock-down NOX2 proteinexpression, to inhibit NADPH oxidase enzyme activity, to effectivelyreduce ROS levels and oxidative stress in atrial tissues at the sites ofinjection, to reduce or altogether eliminate the presence of AF in asubject, or to reduce progression of fibrosis leading to increased AFpresentation in a subject.

NAPDH oxidase (NOX2) is significantly elevated in the ventricle of heartfailure patients, and is thought be a contributor to the heart failurestate. Accordingly, NOX2 shRNAs or expression vectors encoding NOX2shRNAs can be directly injected into ventricle tissue to effectivelyknock-down NOX2 protein expression, to inhibit NADPH oxidase enzymeactivity, to effectively reduce ROS levels and oxidative stress inventricle tissues at the sites of injection, to reduce or altogethereliminate progression of ventricle damage leading to increased HFpresentation in a subject.

In one aspect of the invention, one or more vectors comprising one ormore of shRNA of the invention can be readministered an unlimited numberof times after a first administration at any time interval or intervalsafter the first administration.

shRNA: Pharmaceutical Compositions

The shRNA encoding nucleic acids of the present invention can beformulated in pharmaceutical compositions, which are prepared accordingto conventional pharmaceutical compounding techniques. See, e.g.,Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack PublishingCo., Easton, Pa.). The pharmaceutical compositions of the inventioncomprise a therapeutically effective amount of the vector encodingshRNA. These compositions can comprise, in addition to the vector, apharmaceutically acceptable excipient, carrier, buffer, stabilizer orother materials well known in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The carrier can take a wide variety of forms depending onthe form of preparation desired for administration, e.g., intravenous,oral, intramuscular, subcutaneous, intrathecal, epineural or parenteral.

When the vectors of the invention are prepared for administration, theymay be combined with a pharmaceutically acceptable carrier, diluent orexcipient to form a pharmaceutical formulation, or unit dosage form. Thetotal active ingredients in such formulations include from 0.1 to 99.9%by weight of the formulation

In another aspect of the invention, the vectors of the invention can besuitably formulated and introduced into the environment of the cell byany means that allows for a sufficient portion of the sample to enterthe cell to induce gene silencing, if it is to occur. Many formulationsfor vectors are known in the art and can be used so long as the vectorsgain entry to the target cells so that it can act.

For example, the vectors can be formulated in buffer solutions such asphosphate buffered saline solutions comprising liposomes, micellarstructures, and capsids. The pharmaceutical formulations of the vectorsof the invention can also take the form of an aqueous or anhydroussolution or dispersion, or alternatively the form of an emulsion orsuspension. The pharmaceutical formulations of the vectors of thepresent invention may include, as optional ingredients, solubilizing oremulsifying agents, and salts of the type that are well-known in theart. Specific non-limiting examples of the carriers and/or diluents thatare useful in the pharmaceutical formulations of the present inventioninclude water and physiologically acceptable saline solutions. Otherpharmaceutically acceptable carriers for preparing a composition foradministration to an individual include, for example, solvents orvehicles such as glycols, glycerol, or injectable organic esters. Apharmaceutically acceptable carrier can contain physiologicallyacceptable compounds that act, for example, to stabilize or to increasethe absorption of the shRNA encoding vector. Other physiologicallyacceptable carriers include, for example, carbohydrates, such asglucose, sucrose or dextrans, antioxidants, such as ascorbic acid orglutathione, chelating agents, low molecular weight proteins or otherstabilizers or excipients, saline, dextrose solutions, fructosesolutions, ethanol, or oils of animal, vegetative or synthetic origin.The carrier can also contain other ingredients, for example,preservatives.

It will be recognized that the choice of a pharmaceutically acceptablecarrier, including a physiologically acceptable compound, depends, forexample, on the route of administration of the composition. Thecomposition containing the vectors can also contain a second reagentsuch as a diagnostic reagent, nutritional substance, toxin, oradditional therapeutic agent. Many agents useful in the treatment ofcardiac disease are known in the art and are envisioned for use inconjunction with the vectors of this invention.

Formulations of vectors with cationic lipids can be used to facilitatetransfection of the vectors into cells. For example, cationic lipids,such as lipofectin, cationic glycerol derivatives, and polycationicmolecules, such as polylysine, can be used. Suitable lipids include, forexample, Oligofectamine and Lipofectamine (Life Technologies), which canbe used according to the manufacturer's instructions.

Suitable amounts of vector must be introduced and these amounts can beempirically determined using standard methods. Typically, effectiveconcentrations of individual vector species in the environment of a cellwill be about 50 nanomolar or less 10 nanomolar or less, or compositionsin which concentrations of about 1 nanomolar or less can be used. Inother aspects, the methods utilize a concentration of about 200picomolar or less and even a concentration of about 50 picomolar or lesscan be used in many circumstances. One of skill in the art can determinethe effective concentration for any particular mammalian subject usingstandard methods.

The shRNA is preferably administered in a therapeutically effectiveamount. The actual amount administered, and the rate and time-course ofadministration, will depend on the nature and severity of the condition,disease or disorder being treated. Prescription of treatment, forexample, decisions on dosage, timing, etc., is within the responsibilityof general practitioners or specialists, and typically takes account ofthe disorder, condition or disease to be treated, the condition of theindividual mammalian subject, the site of delivery, the method ofadministration and other factors known to practitioners. Examples oftechniques and protocols can be found in Remington's PharmaceuticalSciences 18th Ed. (1990, Mack Publishing Co., Easton, Pa.).

Alternatively, targeting therapies can be used to deliver the shRNAencoding vectors more specifically to certain types of cell, by the useof targeting systems such as antibodies or cell specific ligands.Targeting can be desirable for a variety of reasons, e.g., if the agentis unacceptably toxic, or if it would otherwise require too high adosage, or if it would not otherwise be able to enter the target cells.

shRNA: Gene Therapy

shRNA can also be delivered into mammalian cells, particularly humancells, by a gene therapy approach, using a DNA vector from which shRNAcompounds in, e.g., small hairpin form (shRNA), can be transcribeddirectly. Recent studies have demonstrated that while double-strandedshRNAs are very effective at mediating RNAi, short, single-stranded,hairpin-shaped RNAs can also mediate RNAi, presumably because they foldinto intramolecular duplexes that are processed into double-strandedshRNAs by cellular enzymes. This discovery has significant andfar-reaching implications, since the production of such shRNAs can bereadily achieved in vivo by transfecting cells or tissues with DNAvectors bearing short inverted repeats separated by a small number of(e.g., 3, 4, 5, 6, 7, 8, 9) nucleotides that direct the transcription ofsuch small hairpin RNAs. Additionally, if mechanisms are included todirect the integration of the vector or a vector segment into thehost-cell genome, or to ensure the stability of the transcriptionvector, the RNAi caused by the encoded shRNAs, can be made stable andheritable. Not only have such techniques been used to “knock down” theexpression of specific genes in mammalian cells, but they have now beensuccessfully employed to knock down the expression of exogenouslyexpressed transgenes, as well as endogenous genes in the brain and liverof living mice.

Gene therapy is carried out according to generally accepted methods asare known in the art. See, for example, U.S. Pat. Nos. 5,837,492 and5,800,998 and references cited therein. Vectors in the context of genetherapy are meant to include those polynucleotide sequences containingsequences sufficient to express a polynucleotide encoded therein. If thepolynucleotide encodes an shRNA, expression will produce the antisensepolynucleotide sequence. Thus, in this context, expression does notrequire that a protein product be synthesized. In addition to the shRNAencoded in the vector, the vector also contains a promoter functional ineukaryotic cells. The shRNA sequence is under control of this promoter.Suitable eukaryotic promoters include those described elsewhere hereinand as are known in the art. The expression vector may also includesequences, such as selectable markers, reporter genes and otherregulatory sequences conventionally used.

Accordingly, the amount of shRNA generated in situ is regulated bycontrolling such factors as the nature of the promoter used to directtranscription of the nucleic acid sequence, (i.e., whether the promoteris constitutive or regulatable, strong or weak) and the number of copiesof the nucleic acid sequence encoding a shRNA sequence that are in thecell. Exemplary promoters include those recognized by pol I, pol II andpol III. In some aspects, a preferred promoter is a pol III promoter,such as the U6 pol III promoter. An exemplary vector for encoding anNOX2 shRNA is depicted in FIG. 4 and illustrated in Table 2.

TABLE 2 Expression vector encoding a Nox2 shRNA. 5′ → 3′ (nucleotide sequence) [SEQ ID NO: 2]aatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcgatcacgagactagcctcgagcggccgcccccttcaccgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgatttcttggctttatatatcttgtggaaaggacgaaacaccggtacaacagccacaacgtctatctcgagatagacgttgtggctgttgtatttttgaattctcgacctcgagacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccactttggccgcggctcgagggggttggggttgcgccttttccaaggcagccctgggtttgcgcagggacgcggctgctctgggcgtggttccgggaaacgcagcggcgccgaccctgggtctcgcacattcttcacgtccgttcgcagcgtcacccggatcttcgccgctacccttgtgggccccccggcgacgcttcctgctccgcccctaagtcgggaaggttccttgcggttcgcggcgtgccggacgtgacaaacggaagccgcacgtctcactagtaccctcgcagacggacagcgccagggagcaatggcagcgcgccgaccgcgatgggctgtggccaatagcggctgctcagcagggcgcgccgagagcagcggccgggaaggggcggtgcgggaggcggggtgtggggcggtagtgtgggccctgttcctgcccgcgcggtgttccgcattctgcaagcctccggagcgcacgtcggcagtcggctccctcgttgaccgaatcaccgacctctctccccagggggatccaccggagcttaccatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacactgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcagtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgacgcccgccccacgacccgcagcgcccgaccgaaaggagcgcacgaccccatgcatcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctaggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgccccactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacctcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagctt

Kits

The subject invention also includes kits for inhibiting expression of atarget gene in a cell, the kit including a chemically modified shRNA asdescribed herein. A “kit” refers to any system for delivering materialsor reagents for carrying out a method of the invention. In the contextof reaction assays, such delivery systems include systems that allow forthe storage, transport, or delivery of reaction reagents (e.g.,chemically modified shRNA, culture medium, etc. in the appropriatecontainers) and/or supporting materials (e.g., buffers, writteninstructions for performing the assay, etc.) from one location toanother. For example, kits include one or more enclosures (e.g., boxes)containing the relevant reaction reagents and/or supporting materials.Such contents may be delivered to the intended recipient together orseparately. For example, a first container may contain a chemicallymodified shRNA for use in an assay, while a second container containsculture media RNA delivery agents (e.g., transfection reagents).

As noted above, the subject kits can further include instructions forusing the components of the kit to practice the subject methods. Theinstructions for practicing the subject methods are generally recordedon a suitable recording medium. For example, the instructions may beprinted on a substrate, such as paper or plastic, etc. As such, theinstructions may be present in the kits as a package insert, in thelabeling of the container of the kit or components thereof (i.e.,associated with the packaging or subpackaging) etc. In otherembodiments, the instructions are present as an electronic storage datafile present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate.

In addition to the subject database, programming and instructions, thekits may also include one or more control reagents, e.g., non-chemicallymodified shRNA.

Methods

The pharmaceutical compositions have therapeutic efficacy in treatingoxidative stress in a subject having atrial or ventricular arrhythmias,ventricular failure or heart failure. A pharmaceutical compositioncomprising a NOX2 shRNA has demonstrable activity in an art-acceptedcanine model for human AF. Experiments were performed with normal and HFanimals to validate the utility of the canine model to study NOX2knockdown assays and their effect on NOX2 expression and superoxideproduction. The HF animals produced superoxide at higher levels in theposterior left atrium (PLA) than in the left atrial appendage (LAA) ascompared to normal animals (FIG. 2A). NADPH oxidase is a major source ofsuperoxide generation in PLA from HF animals compared to normal animals(FIG. 2B) depicts contribution of O₂ ⁻ production in PLA for NADPHoxidase (blue bars), Mitochondrial ROS (red bars), NOS (green bars) andXanthine oxidase (purple bars). Furthermore, HF animals display higherlevels of NOX2 polypeptide subunit (gp91) as compared to normal animals(FIG. 3A, B).

A plasmid encoding either NOX2 shRNA (FIG. 4 and SEQ ID NOs: 1 and 2) ora control RNA (lac Z) was introduced into PLA tissues of HF animals andthe effects on AF were evaluated. The duration of AF (seconds) isdecreased in plasmid encoding NOX2 shRNA-treated animals (“NOX2 shRNA”)compared with lacZ-transfected control animals (“HF (LacZ Control)”)(FIG. 5). Moreover, expression of ox-CAMKII in the PLA of HF dogstransfected with NOX2 shRNA (FIG. 6B) is lower than expression ofox-CAMKII in the PLA of HF dogs (FIG. 6A), which is consistent with NOX2shRNA knocking down NOX2 expression and lowering oxidative stress inatrial tissues.

Electrophysiological measurements were recorded for animals following 3weeks of rapid atrial pacing. Effective Refractory Periods (ERPs) wereincreased significantly following pacing conditioning; however, ERPs didnot change significantly as a function of NOX2 knockdown using NOX2shRNA (FIG. 7). Conduction was less heterogeneous in PLA transfectedwith plasmid encoding NOX2 shRNA compared to PLA transfected withcontrol plasmid (FIG. 8A, B) and NOX2 shRNA abolished significant changenoted in the conductive inhomogeneity index (FIG. 8C, D).

NOX2 mRNA and protein is reduced in PLA transfected with plasmidencoding NOX2 shRNA compared to PLA transfected with control plasmid(FIG. 9A, B). Similarly, the relative contribution of superoxideproduction attributed to NADPH oxidase is lower in PLA transfected withplasmid encoding NOX2 shRNA compared to PLA transfected with controlplasmid (FIG. 10).

In addition to using traditional heart monitoring methodologies todetect the presence and absence of AF as a function of NOX2 shRNAtherapy, AF EGM characteristics can be systematically assessed toevaluate structural remodeling of atrial tissue in candidate subjects aswell as therapeutic efficacy of the pharmaceutical compositions forinhibiting oxidative stress for subjects suffering from chronic AF.Subjects who suffer from chronic AF have structural remodeled atrialtissue with fibrosis. In this regard, fibrosis was reduced in animalswhose PLA was transfected with plasmid encoding NOX2 shRNA compared tocontrol animals (FIG. 11A-C).

Application of EGM-guided identification and monitoring offibrosis-associated structural remodeling of atrial tissue in AF and HFis summarized by related patent application publications by theinventors, such as application entitled “INHIBITION OF FIBROSIS AND AFBY TGF-BETA INHIBITION IN THE POSTERIOR LEFT ATRIUM (PLA)” to RishiArora, bearing Ser. No. 13/890,116, filed May 8, 2013, now U.S. PatentApplication Publication US 2014-0037545 A1 and application entitled“USING INTRACARDIAC ELECTROGRAMS TO PREDICT LOCATION OF FIBROSIS ANDAUTONOMIC NERVES IN THE HEART” to Rishi Arora et at., bearing Ser. No.13/890,112, filed May 8, 2013, now U.S. Patent Application PublicationUS 2013-0324869A1.

In another model, electrical remodeling of atrial tissues can lead tospontaneous AF. Such a model reflects AF in a subject who otherwisemight not have significant structural remodeling of atrial tissue due tofibrosis (for example, young subjects). Pacemakers can be used togenerate rapid tachypacing in the canine model of electrical remodelingof atrial tissue. Heart monitors (for example, ECG or EKG patterns) canbe used to detect AF development as well as the efficacy of NOX2 shRNAtreatment efficacy in atrial tissues by measuring the decline or absenceof AF following treatment. In this regard, the onset of AF episodes wasblocked in animals whose PLA was transfected with plasmid encoding NOX2shRNA, as compared to normal animals following rapid atrial pacing ofthe animals (FIG. 12). Applicability to other NOX2 inhibitors.

The results of the NOX2 shRNA studies demonstrate the feasibility of ageneral strategy to inhibit NADPH oxidase by targeting NOX2 using NOX2inhibitors. Such inhibitor agents include oligonucleotide-basedcompounds that target the NOX2 mRNA or protein, such as RNAi moleculesand shRNAs directed against NOX2 mRNA and oligonucleotide-based aptamersdirected against the NOX2 polypeptide. Furthermore, small moleculeorganic compounds having anti-NADPH oxidase activity by specificallybinding to or otherwise interfering with NOX2 protein functionality canalso reduce NADPH oxidase-mediated superoxide production and oxidativestress in AF.

In some embodiments, NOX2 inhibitors comprise any suitable bioactivemolecules (e.g. a molecule capable of inhibiting the function of NADPHoxidase). In some embodiments, a NOX2 inhibitor comprises amacromolecule, polymer, a molecular complex, protein, peptide,polypeptide, nucleic acid, carbohydrate, small molecule, etc.

In some embodiments, a NOX2 inhibitor is a NOX2 inhibitory peptide. Insome embodiments, the present invention provides peptides of anysuitable amino acid sequence capable of inhibiting one or more allelesof NOX2. In some embodiments, peptides provided by or encoded by thecompositions of embodiments of the present invention may comprise anyarrangement of any standard amino acids (e.g. alanine, arginine,asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,proline, serine, threonine, tryptophan, tyrosine, and valine) ornon-standard amino acids (e.g. D-amino acids, chemically or biologicallyproduced derivatives of common amino acids, selenocysteine, pyrrolysine,lanthionine, 2-aminoisobutyric acid, dehydroalanine, etc.). In someembodiments, NOX2 inhibitory peptides are inhibitors to NOX2.

In some embodiments, NOX2 inhibitory peptides are provided to a subjectas isolated or purified peptides. In some embodiments, NOX2 inhibitorypeptides are provided to a subject as nucleic acid molecules that encodesuch peptides. In some embodiments, peptides are optimized to enhancecell penetration (e.g. sequence optimization, sequence tag, tagged witha small molecule, etc.).

In some embodiments, a NOX2 inhibitor is provided from an isolatednucleic acid comprising a minigene, wherein said minigene encodes amodified NOX2 peptide, wherein the peptide blocks the site ofinteraction between NOX2 and NOX2 binding partners in a cell, such as ahuman cell. In addition, the minigene can further comprise one or moreof a promoter, a ribosomal binding site, a translation initiation codon,and a translation termination codon. In some embodiments, the minigeneencodes a modified NOX2. peptide having one of the following generalformulas: MGX, MX, and MZX, wherein M is a methionine amino acidresidue, wherein G is a glycine amino acid residue, wherein Z is anamino acid residue other than a glycine amino acid residue, and whereinX is a NOX2. peptide which comprises an amino acid sequence from theNOX2. subunit, and has the property of binding a NOX2 binding partner.In this embodiment, X can comprise from at least about three contiguousamino acids to at least about 54 contiguous amino acids, from at leastabout three contiguous amino acids to at least about eleven contiguousamino acids, and at least about eleven contiguous amino acids. In oneembodiment, X comprises the seven contiguous amino acid residues of theNOX2 subunit.

In some embodiments, the NOX2 inhibitor is provided as an isolated orpurified polypeptide. In some embodiments, the peptide has a generalformula selected from the group consisting of MGX, MX, and MZX, whereinM is a methionine amino acid residue, wherein G is a glycine amino acidresidue, wherein Z is an amino acid residue other than a glycine aminoacid residue, and wherein X is a NOX2-derived peptide which comprises anamino acid sequence of the NOX2. subunit, and has the property ofbinding a NOX2 binding partner. In this embodiment, X can comprise fromat least about three contiguous amino acids to at least about 54contiguous amino acids, from at least about three contiguous amino acidsto at least about eleven contiguous amino acids, and at least abouteleven contiguous amino acids. In one embodiment, X comprises the sevencontiguous amino acid residues of the NOX2 subunit.

In some embodiments, the present invention provides methods ofinhibiting a NOX2-mediated signaling event in a cell or tissue. Thesemethods comprise administering to a cell or tissue, preferably a humancell or tissue, one of a modified NOX2 peptide and an isolated nucleicacid comprising a minigene which encodes a modified NOX2 peptide,whereby following the administration, the NOX2 peptide inhibits theNOX2-mediated signaling event in the cell or tissue.

In some embodiments, a NOX2 inhibitor comprises a small molecule. Insome embodiments, the present invention provides a small moleculeinhibitor of NOX2. In some embodiments, the present invention provides asmall molecule drug or pharmaceutical compound configured to or capableof inhibiting NOX2 activity, function expression, or the like.

In some embodiments, the present invention provides RNAi molecules(e.g., that alter NOX2 expression) as a NOX2 inhibitor. In someembodiments, the present invention targets the expression of NOX2 genesusing nucleic acid based therapies. For example, in some embodiments,the present invention employs compositions comprising oligomericantisense or RNAi compounds, particularly oligonucleotides, for use inmodulating the function of nucleic acid molecules encoding NOX2 genes,ultimately modulating the amount of NOX2 protein expressed. In someembodiments, RNAi is utilized to inhibit NOX2 gene function. RNAirepresents an evolutionary conserved cellular defense for controllingthe expression of foreign genes in most eukaryotes, including humans.RNAi is typically triggered by double-stranded RNA (dsRNA) and causessequence-specific mRNA degradation of single-stranded target RNAshomologous in response to dsRNA. The mediators of mRNA degradation aresmall interfering RNA duplexes (siRNAs), which are normally producedfrom long dsRNA by enzymatic cleavage in the cell. siRNAs are generallyapproximately twenty-one nucleotides in length (e.g. 21-23 nucleotidesin length), and have a base-paired structure characterized by twonucleotide 3′-overhangs. Following the introduction of a small RNA, orRNAi, into the cell, it is believed the sequence is delivered to anenzyme complex called RISC (RNA-induced silencing complex). RISCrecognizes the target and cleaves it with an endonuclease. It is notedthat if larger RNA sequences are delivered to a cell, RNase III enzyme(Dicer) converts longer dsRNA into 21-23 nt ds siRNA fragments.

The transfection of siRNAs into animal cells results in the potent,long-lasting post-transcriptional silencing of specific genes (Caplen etal, Proc Natl Acad Sci U.S.A. 2001; 98: 9742-7; Elbashir et al., Nature.2001; 411:494-8; Elbashir et al., Genes Dev. 2001; 15: 188-200; andElbashir et al., EMBO J. 2001; 20: 6877-88, all of which are hereinincorporated by reference). Methods and compositions for performing RNAiwith siRNAs are described, for example, in U.S. Pat. No. 6,506,559,herein incorporated by reference.

siRNAs are extraordinarily effective at lowering the amounts of targetedRNA, and by extension proteins, frequently to undetectable levels. Thesilencing effect can last several months, and is extraordinarilyspecific, because one nucleotide mismatch between the target RNA and thecentral region of the siRNA is frequently sufficient to preventsilencing (Brummelkamp et al, Science 2002; 296:550-3; and Holen et al,Nucleic Acids Res. 2002; 30:1757-66, both of which are hereinincorporated by reference).

In some embodiments, NOX2 expression is modulated using antisensecompounds that specifically hybridize with one or more nucleic acidsencoding NOX2. The specific hybridization of an oligomeric compound withits target nucleic acid interferes with the normal function of thenucleic acid. This modulation of function of a target nucleic acid bycompounds that specifically hybridize to it is generally referred to as“antisense.”

In some embodiments, the present invention contemplates the use of anygenetic manipulation for use in modulating the expression of NOX2 genes.Examples of genetic manipulation include, but are not limited to, geneknockout (e.g., removing the NOX2 gene from the chromosome using, forexample, recombination), expression of antisense constructs with orwithout inducible promoters, and the like. Delivery of nucleic acidconstruct to cells in vitro or in vivo may be conducted using anysuitable method. A suitable method is one that introduces the nucleicacid construct into the cell such that the desired event occurs (e.g.,expression of an antisense construct). Genetic therapy may also be usedto deliver siRNA or other interfering molecules that are expressed invivo (e.g., upon stimulation by an inducible promoter.

In some embodiments, the present invention provides antibodies thattarget NOX2 protein. Any suitable antibody (e.g., monoclonal,polyclonal, or synthetic) may be utilized in the therapeutic methodsdisclosed herein. In preferred embodiments, the antibodies are humanizedantibodies. Methods for humanizing antibodies are well known in the art(See e.g., U.S. Pat. Nos. 6,180,370, 5,585,089, 6,054,297, and5,565,332; each of which is herein incorporated by reference).

In some embodiments, the present invention provides methods of enhancingentry of a NOX2 inhibitor into cells or tissue. In some embodiments, thepresent invention provides administering a NOX2 inhibitor in conjunctionwith electroporation, electropermeabilization, or sonoporation. In someembodiments, the present invention provides administering a NOX2inhibitor in conjunction with electroporation. In some embodiments, thepresent invention provides co-injection/electroporation of the tissue ofa subject. In some embodiments, the present invention providesadministering a NOX2 inhibitor prior to, simultaneously with, and/orfollowing electroporation. In some embodiments, electroporation providesa method of delivering pharmaceuticals or nucleic acids (e.g. DNA) intocells. In some embodiments, tissue electrically stimulated at the sametime or shortly after pharmaceutical or DNA is applied (e.g. NOX2inhibitor). In some embodiments, electroporation increases cellpermeability. The permeability or the pores are large enough to allowthe pharmaceuticals and/or DNA to gain access to the cells. In someembodiments, the pores in the cell membrane close and the cell onceagain becomes impermeable or less permeable. Devices forco-injection/electroporation are known in the art (U.S. Pat. No.7,328,064, herein incorporated by reference in its entirety).

Furthermore, though the canine model utilized PLA as model atrialtissue, the approach is applicable to all atrial tissues. In someembodiments, the present invention provides compositions and methods totreat or prevent conditions and/or diseases of the heart (e.g. rhythmdisturbances (e.g. atrial fibrillation)). In some embodiments, thepresent invention provides treatment or prevention of a heart disease orcondition selected from the list of aortic dissection, cardiacarrhythmia (e.g. atrial cardiac arrhythmia (e.g. premature atrialcontractions, wandering atrial pacemaker, multifocal atrial tachycardia,atrial flutter, atrial fibrillation, etc.), junctional arrhythmias (e.g.supraventricular tachycardia, AV nodal reentrant tachycardia, paroxysmalsupra-ventricular tachycardia, junctional rhythm, junctionaltachycardia, premature junctional complex, etc.), atrio-ventriculararrhythmias, ventricular arrhythmias (e.g. premature ventricularcontractions, accelerated idioventricular rhythm, monomorphicventricular tachycardia, polymorphic ventricular tachycardia,ventricular fibrillation, etc.), etc.), congenital heart disease,myocardial infarction, dilated cardiomyopathy, hypertrophiccardiomyopathy, aortic regurgitation, aortic stenosis, mitralregurgitation, mitral stenosis, Ellis-van Creveld syndrome, familialhypertrophic cardiomyopathy, Holt-Orams Syndrome, Marfan Syndrome,Ward-Romano Syndrome, and/or similar diseases and conditions.

In some embodiments, the present invention provides compositions andmethods to treat atrial fibrillation. Atrial fibrillation is thecommonest rhythm disturbance of the heart. The posterior left atrium andpulmonary veins have been shown to play an important role in the genesisof atrial fibrillation. More recent studies demonstrate a role for theautonomic nervous system, especially the parasympathetic nervous system,in the genesis of atrial fibrillation from the posterior left atrium.Current therapies to manage atrial fibrillation remain ineffective,while novel links, including autonomic activity described here, providefor beneficial treatment options. Work conducted during the developmentof embodiments of the present invention shows that selective disruptionof autonomic pathways in the posterior left atrium can significantlymodify the ability to the heart to sustain atrial fibrillation. Inparticular, embodiments of the present invention treat atrialfibrillation by administration of NOX2 inhibitors. An understanding ofthe mechanism of action is not necessary to practice the presentinvention and the present invention is not limited to any particularmechanism of action. The present invention also provides compositionsand methods for researching atrial fibrillation, including screening forcompounds useful in treating, prevent, or reducing signs or symptomsassociate with atrial fibrillation.

EXAMPLES

The invention will be more fully understood upon consideration of thefollowing non-limiting examples, which are offered for purposes ofillustration, not limitation.

Example 1. HF Canine Model of Structural Remodeling and AF

Dog Protocols.

Purpose-bred hound dogs (weight range: 25-35 kg; age rage: 1-3 years)used in this study were maintained in accordance to the Guide for theCare and Use of Laboratory Animals published by the U.S. NationalInstitutes of Health (NIH Publication No. 85-23, revised 1996) asapproved by the Animal Care and Use Committee of NorthwesternUniversity. Before undergoing the procedures listed below, all animalswere sedated with diazepam 0.22 mg/kg IM and were induced with propofol(3-7 mg/kg IV). After animals were unresponsive, they were intubated andpositive-pressure ventilated, and anesthesia was maintained withisoflurane 0.5-1.5%. The surgical field was scrubbed with chlorhexidineand isopropyl alcohol, sprayed with betadine solution, and draped withsterile towels and sheets. The chest was then opened via a left lateralthoracotomy.

Eleven hounds were randomized into 2 groups: 1) control (n=7) expressingLacZ and 2) gene therapy with a plasmid expressing NOX2 shRNA (n=4).During an initial procedure, a left lateral thoracotomy was performedand the pericardium was incised; animals underwent invasiveelectrophysiology study (EPS), gene injection, echocardiogram andepicardial implantation of a left ventricular pacemaker. After animalswere allowed to recover for 3-5 days, ventricular tachypacing wasinitiated at 240 bpm. Clinical status assessment and pacing was verifieddaily. Three weeks after the initial procedure, animals underwentterminal echocardiogram, FPS and cardiac extraction for histology andmolecular studies.

Initial Open Chest Electrophysiological Mapping

Effective Refractory Periods (ERPs). ERPs were obtained using tworectangular plaques epicaridally positioned on the PLA and LAA. Eachplaque had 21 electrodes each (7×3 electrodes, inter-electrodedistance=5 mm). A Bloom stimulator interfaced with a GE Prucka Cardiolabsystem (GE Healthcare, Waukesha, Wis.) were used to perform programmedstimulation. ERPs were measured by delivering an 8 pulse drive train at400 ms coupled to a premature stimulus. The coupling interval of thepremature stimulus was decreased in 10 ms increments, and the ERP wasdefined as the longest coupling interval that fails to conduct. ERPswere obtained from five distributed sites in the both the PLA and LAA.FIG. 7 depicts changes in ERP in plasmid encoding NOX2 shRNA-treatedanimals (NOX2 shRNA) compared with lacZ-transfected control animals (HF)for baseline (black bars) and post-pacing (grey bars) conditionedanimals.

AF induciblility. AF was induced with burst pacing at maximum outputusing cycle lengths of 180 ms to 100 ms with 10 ms decrements for 10seconds for each cycle length. Current was set at four times thresholdfor capture. AF induction was defined as episodes lasting more than 30seconds.

Activation Mapping. High density activation mapping was performed usinga UNEMAP mapping system (Univ. of Auckland, Auckland, New Zealand). Atriangular plaque containing 130 electrodes (inter-electrode distance of2.5 mm) were used to record 117 bipolar EGMs at a 1 kHz sampling rate.Mapping was performed sequentially in the LAA and in two adjacent sitesin the PLA. At each site, 10 second recordings were made during sinusrhythm and pacing with cycle lengths of 400, 300, and 200 ms. Pacing wasperformed in LAA when recording from the PLA and vice versa.

Data analysis with Conduction Inhomogeneity. MATLAB (Mathworks, Natick,Mass.) was used for all offline signal analysis in this study.Conduction inhomogeneity analysis was performed using the high densityUNEMAP recordings obtained during sinus rhythm and pacing. The bipolarelectrograms were highpass filtered at 30 Hz, rectified, and thenlowpass filtered at 20 Hz. The times of the filtering peaks wereconsidered the activation time for that activation. Conductioninhomogeneity was calculated as described by Lammers W J, Schalij M J,Kirchhof C J, Allessie M A. Quantification of spatial inhomogeneity inconduction and initiation of reentrant atrial arrhythmias. Am J Physiol1990; 259:H1254-63. Total activation time measuring the amount of timeto activate the entire area of the plaque was also measured. Totalactivation time measuring the amount of time to activate the entire areaof the plaque was also measured. Exemplary results of these studies areshown in FIG. 8A-D.

Gene Injection/Transfer.

After the completion of the initial FPS, 10 mg of plasmid DNA [controlplasmid (pUBc-lacZ) or NOX2 shRNA-encoded plasmid expression vectorunder the control of the U6 pol III promoter (FIG. 4)] (0.1 mg/eachsite) was directly injected transmurally in the PLA with a 27-gaugeneedle. Electric pulses were delivered to the myocardium through theelectrodes spaced 1 cm apart (Genetrodes, BTX). Immediately after theinjection, 8 electrical pulses (amplitude, 200 V; duration, 10 ms;intervals, 1 sec) were delivered with a square-wave electroporationgenerator (ECM 830, BTX, Harvard Apparatus).

HF Induction.

In 11 dogs, HF was induced by 3 to 4 weeks of right ventriculartachypacing (240 beats per minute) by an implanted pacemaker. In alldogs, a pacemaker was placed on the ventricle via an epicardial approach(that is, via a left lateral thoracotomy). Left ventricular function wasassessed during pacing by serial echocardiograms (data not shown). HFwas confirmed after 3 to 4 weeks of pacing.

Terminal surgery and open chest electrophysiology mapping. At theterminal study, a left lateral thoracotomy was performed. Low densityand high density mapping protocols were used. With low density mapping,the posterior left atrium (PLA), left superior pulmonary vein (PV), andleft atrial appendage could be mapped simultaneously. The PLA and LAAwere mapped using two rectangular plaques containing 21 electrodes each(7×3 electrodes, inter-electrode distance=5 mm) from which 18 bipolarEGMs were recorded. FIG. 3A shows the schematics of the plaques. Thesignals from the low density plaques were recorded and stored at a 977Hz sampling rate with the GE Prucka Cardiolab system (GE Healthcare,Waukesha, Wis.).

High density mapping was performed in all dogs for more detailedelectrophysiological analysis, including assess of conductionheterogeneity (inhomogeneity). Mapping was performed sequentially in thePLA and LAA with a triangular plaque containing 130 electrodes(inter-electrode distance of 2.5 mm) from which 117 bipolar electrograms(EGMs) were recorded. The UNEMAP mapping system (Univ. of Auckland,Auckland, New Zealand) was used for recording and storing the EGMs at a1 kHz sampling rate. Even though we did not separately map the PVsduring high-density mapping (owing to the relatively large surface areaof the high density plaques, it was technically challenging to cover thePVs, which have a circular and uneven surface), the high-density plaquedid straddle the proximal PVs during PLA mapping.

Example 2. Rapid Atrial Pacing (RAP) Canine Model of ElectricalRemodeling and Spontaneous AF

A transvenous pacemaker was placed in the right atrium (RA) via ajugular venous approach. One week later, the animal underwent a leftlateral thoracotomy. Open chest electrophysiological mapping wasperformed as follows:

Initial Open Chest Electrophysiological Mapping

Effective Refractory Periods (ERPs). ERPs were obtained using tworectangular, 21-electrode plaques epicardially positioned on the PLA andLAA. ERPs were obtained from five evenly distributed sites in the boththe PLA and LAA.

AF induciblility. AF was induced with burst pacing. AF was defined asepisodes lasting more than 3 seconds. Sustained AF was defined as AF>60seconds.

Activation Mapping. High density activation mapping was performed usingthe UNEMAP mapping system (Univ. of Auckland, Auckland, New Zealand). Atriangular plaque containing 130 electrodes (inter-electrode distance of2.5 mm) were used to record 117 bipolar EGMs at a 1 kHz sampling rate.Mapping was performed sequentially in the LAA and in two adjacent sitesin the PLA. At each site, 10 second recordings were made during sinusrhythm and pacing with cycle lengths of 400, 300, and 200 ms.

Gene injection/transfer. After the completion of the initialelectrophysiological study, 10 mg of plasmid [control plasmid(pUBc-lacZ) or NOX2 shRNA-encoded plasmid expression vector under thecontrol of the U6 pol DI promoter (FIG. 4))] (diluted in 5 ml sterilesaline) was injected sub-epicardially in the PLA with a 27-gauge needle;5-6 injections (approximately 1 ml each) were used to cover the entirePLA. After gene injection, electroporation was performed at each site ofgene injection (200 V; duration, 10 ms; intervals, 1 sec).

Rapid atrial pacing. After the initial open-chest mapping study and geneinjection, the chest was closed and the animal allowed to recover for 1week. Rapid atrial pacing (at 600 beats/min) was then started, andcontinued for the next 3 weeks. FIG. 12 illustrates that dog recipientsof NOX2 shRNA-encoded plasmid expression vector injected into PLA tissuedid not develop spontaneous AF following rapid atrial pacing.

Serial testing for sustained AF. Every 24-48 hours, pacing was stoppedfor 30-60 minutes, in order to assess for onset of AF and for durationof the induced AF.

Terminal surgery and open chest electrophysiology mapping. After 3 weeksof rapid atrial pacing (RAP), repeat open chest electrophysiologicalmapping was performed. If the animal reverted to sinus rhythm, atrialeffective refractory periods were assessed. AF electrograms were alsorecorded.

After the electrophysiological study was completed, the heart wasremoved and the atria snap frozen. The atria was subjected to furtheranalysis to assess for gene expression, expression of key signalingmolecules etc.

Example 3. Histology and Tissue Analysis

The histologic analysis described below was performed on atrial tissuesfrom untreated and treated dogs receiving plasmids expressing NOX2 shRNAor an inactive control RNA (lac Z) were analyzed.

Tissue Sample Preparation. In the animals undergoing high densitymapping, immediately following the in vivo electrophysiological study,the heart was promptly excised out of the chest and immersed in ice-coldcardioplegia as previously described by us. After marking the exactorientation of the high density plaques, tissue samples were taken fromthe PLA and LAA regions of the left atrium and snap frozen in liquidnitrogen. Samples were saved in the exact orientation in which highdensity mapping had been performed. All samples were initially saved at−80° C. The oriented tissue samples were frozen in Tissue-Tek OCT(Optimal Cutting Temperature) compound at −80° C.

For paraffinization, the tissue was thawed and a quick wash given toclean off all the OCT. Using a PCF LEICA 1050 Tissue Processor, thetissue was embedded in paraffin. The tissue processor uses 10% NBF(Neutral Buffered Formalin) for fixing and the tissue dehydration isperformed with incremental concentrations of Ethanol (ETOH). ETOH isexchanged with xylene and finally xylene is exchanged with paraffin at58° C. Then tissue is embedded in a paraffin block.

Masson's Trichrome staining. Tissue sections were cut 4 μm apart.Paraffin was removed by placing the tissue section in histology gradexylene for two minutes and the process was repeated four times changingxylene solution after every two minutes. Finally, the xylene was washedaway with ETOH for one minute in absolute ETOH, then again fir one moreminute with fresh absolute ETOH, followed by wash in 95% ETOH for 30seconds, and subsequently in 70% ETOH for 45 seconds. ETOH was thenwashed with water for one minute. The tissue section was then ready forstaining. The section was treated with Bouin's mordant at roomtemperature overnight. The following day the tissue section was rinsedin running water to remove excess yellow. The tissue section was stainedin Weigert's Solution for 7 minutes. Next, it was dipped once in 1% acidalcohol and immediately rinsed. The section was then stained inBeibrich. Scarlet-Acid fuchsin for 2 minutes, followed by a rinse indistilled water. Subsequently, the tissue section was stained inphosphomolybdic-phosphotungstic acid solution for 6 minutes, followed byanother rinse in distilled water. The issue section was then stained inAniline Blue solution for 5 minutes, followed by another rinse indistilled water. Immediately, the tissue was dipped once in 1% Glacialacetic acid and quickly rinsed. The tissue section was then dehydratedin twice in each concentration of 95% and 100% of ETOH, which was laterexchanged with xylene. A coverslip was finally placed on the tissuesection for microscope examination. Examples of results of such assaysare illustrated in FIG. 11A-C.

ox-CAMKII immunofluorescence. Tissue samples were fixed and stained withanti-ox-CAMKII antibody. Exemplary results of such assays areillustrated in FIG. 6A, B.

Real-time PCR. Frozen tissue samples were frozen crushed andhomogenized. Total RNA was isolated using Trizol Reagent (LifeTechnologies, 15596-026). Contaminating DNA was removed using

DNA-free DNA Removal Kit (Life Technologies, AM1906). cDNA wassynthesized from 0.5 μg of total RNA with TaqMan reverse transcriptionreagents SuperScript VILO (Life Technologies, #11755050) and mixed withTaqMan Fast Advanced Master Mix (Life Technologies, #444965).Quantitative real-time PCR (qRT-PCR) was carried out using AppliedBiosystems® 7500 Fast Real-Time PCR System (Life Technologies). RelativemRNA levels were calculated by the (type of software) afternormalization of each experimental sample to GAPDH levels. An example ofresults of such assays is illustrated in FIG. 9A.

Western Blot Analysis. The atrial tissue was snap frozen in liquidnitrogen, homogenized, and separated into membrane and cytosolicfractions according to the procedures of the Mem-Per Plus MembraneProtein Extraction Kit (Thermo-Scientific, #89842) or T-Per TissueProtein Extraction Reagent (Thermo-Scientific, 78510) for total protein.We added Halt Protease & Phosphatase Inhibitor Cocktail(Thermo-Scientific, #78446) to all buffers. Protein concentrations weredetermined using Pierce BCA Protein Assay Kit (Thermo-Scientific,#23227). Proteins were fractionated by SDS-PAGE, transferred topolyvinyl difluoride (PVDF) membrane, blocked with 5% BSA, and blottedwith the appropriate primary and secondary antibodies. Examples ofresults of such assays are illustrated in FIG. 3 and FIG. 9B.

Measurement of NADPH-dependent superoxide production bychemiluminescence. 100 mg frozen tissue samples were crushed and rotorhomogenized (Biospec Products Inc, Tissue-Tearor) with proteaseinhibitor (Halt protease and phosphatase inhibitor cocktail,Thermo-Scientific, #1861282). Protein concentrations were determinedusing Pierce BCA Protein Assay Kit (Thermo-Scientific, #23227).Lucigenin (5 μmol/L) (Enzo Life Sciences, ALX-620-061-M050) and NADPH(100 μmol/L) (Calbiochem, #481973) were each added in the presence andabsence of apocynin (Santa Cruz Biotechnology, #sc-203321) (an inhibitorof NADPH oxidase) and mito-TEMPO (Santa Cruz Biotechnology, #sc-221945)(a mitochondrial ROS inhibitor), and the photon outputs were measuredusing a luminometer (Berthold Technologies, LUMAT LB 9507). An exampleof this assay is described for PLA and LLA tissue samples from normaland HF dogs in FIG. 2A, B.

REFERENCES

-   1. Aistrup G C I, Ng J, Gordon D, Koduri H, Browne S, Arapi D, Segon    Y, Goldstein J, Angulo A, Wasserstrom J A, Goldberger J, Kadish A,    Arora R. Non-viral Gene-based Inhibition of Gi/o-mediated Vagal    Signaling in the Posterior Left Atrium Decreases Vagal Induced A F.    Heart Rhythm 2011; 8:1722-9-   2. Benjamin E J, Levy D, Vaziri S M, D'Agostino R B, Belanger A J,    Wolf P A. Independent risk factors for atrial fibrillation in a    population-based cohort. The Framingham Heart Study. JAMA 1994;    271:840-4-   3. Gerstenfeld F P, Sauer W, Callans D J, et al. Predictors of    success after selective pulmonary vein isolation of arrhythmogenic    pulmonary veins for treatment of atrial fibrillation. Heart Rhythm    2006; 3:165-70-   4. Pappone C, Oral H, Santinelli V, et al. Atrio-esophageal fistula    as a complication of percutaneous transcatheter ablation of atrial    fibrillation. Circulation 2004; 109:2724-6-   5. Verma A, Patel D, Famy T, et al. Efficacy of adjuvant anterior    left atrial ablation during intracardiac echocardiography-guided    pulmonary vein antrum isolation for atrial fibrillation. J    Cardiovasc Electrophysiol 2007; 18:151-6-   6. Rodrigues A C, Scannavacca M I, Caldas M A, et al. Left atrial    function after ablation for paroxysmal atrial fibrillation. American    Journal of Cardiology 2009; 103:395-8-   7. Aldhoon B, Melenovsky V, Peichl P, Kautzner J. New insights into    mechanisms of atrial fibrillation. Physiological research/Academia    Scientiarum Bohemoslovaca 2010; 59:1-12-   8. Jeong E-M, Liu M, Sturdy M, et al. Metabolic stress, reactive    oxygen species, and arrhythmia. Journal of Molecular and Cellular    Cardiology 2012; 52:454-63-   9. Hool L C. Reactive Oxygen Species in Cardiac Signalling: From    Mitochondria to Plasma Membrane Ion Channels. Clinical and    Experimental Pharmacology and Physiology 2006; 33:146-51-   10. Zima A V, Blatter L A. Redox regulation of cardiac calcium    channels and transporters. Cardiovascular Research 2006; 71:310-21-   11. Nediani C, Raimondi L, Borchi E, Cerbai E. Nitric Oxide/Reactive    Oxygen Species Generation and Nitroso/Redox Imbalance in Heart    Failure: From Molecular Mechanisms to Therapeutic Implications.    Antioxidants & Redox Signaling 2011; 14:289-331-   12. Lijnen P J, van Pelt J F, Fagard R H. Stimulation of reactive    oxygen species and collagen synthesis by angiotensin II in cardiac    fibroblasts. Cardiovascular therapeutics 2012; 30:e1-8-   13. Nabeebaccus A, Zhang M, Shah A M. NADPH oxidases and cardiac    remodelling. Heart Fail Rev 2011; 16:5-12-   14. Erickson J R, Joiner M L, Guan X, et al. A dynamic pathway for    calcium-independent activation of CaMKII by methionine oxidation.    Cell 2008; 133:462-74-   15. Youn J-Y, Zhang J, Zhang Y, et al. Oxidative stress in atrial    fibrillation: An emerging role of NADPH oxidase. Journal of    Molecular and Cellular Cardiology 2013; 62:72-9-   16. Murdoch C E, Zhang M, Cave A C, Shah A M. NADPH    oxidase-dependent redox signalling in cardiac hypertrophy,    remodelling and failure. Cardiovasc Res 2006; 71:208-15-   17. Kuroda J, Ago T, Matsushima S, Zhai P, Schneider M D,    Sadoshima J. NADPH oxidase 4 (Nox4) is a major source of oxidative    stress in the failing heart. Proc Natl Acad Sci USA 2010;    107:15565-70-   18. Yeh Y H, Kuo C T, Chang G J, Qi X Y, Nattel S, Chen W J.    Nicotinamide adenine dinucleotide phosphate oxidase 4 mediates the    differential responsiveness of atrial versus ventricular fibroblasts    to transforming growth factor-beta. Circ Arrhythm Electrophysiol    2013; 6: 790-8-   19. Zhang M, Perino A, Ghigo A, Hirsch E, Shah A M. NADPH oxidases    in heart failure: poachers or gamekeepers? Antioxid Redox Signal    2013; 18:1024-41-   20. Reilly S N, Jayaram R, Nahar K, et al. Atrial sources of    reactive oxygen species vary with the duration and substrate of    atrial fibrillation: implications for the antiarrhythmic effect of    statins. Circulation 2011; 124:1107-17-   21. Ciaccio E J, Biviano A B, Whang W, Gambhir A, Garan H. Different    characteristics of complex fractionated atrial electrograms in acute    paroxysmal versus long-standing persistent atrial fibrillation.    Heart Rhythm 2010; 7:1207-15-   22. Ciaccio E J, Biviano A B, Whang W, et al. Differences in    repeating patterns of complex fractionated left atrial electrograms    in longstanding persistent atrial fibrillation as compared with    paroxysmal atrial fibrillation. Circ Arrhythm Electrophysiol 2011;    4:470-7-   23. Aistrup G L, Cokic I, Ng J, et al. Targeted nonviral gene-based    inhibition of Galpha(i/o)-mediated vagal signaling in the posterior    left atrium decreases vagal-induced atrial fibrillation. Heart    Rhythm 2011; 8:1722-9.-   24. Aistrup G L, Villuendas R, Ng J, et al. Targeted G-protein    inhibition as a novel approach to decrease vagal atrial fibrillation    by selective parasympathetic attenuation. Cardiovasc Res 2009;    83:481-92.-   25. Balasubramaniam R, Kistler P M. A F and Heart failure: the    chicken or the egg?. Heart 2009; 95:535-9.-   26 Lakshminarayan K, Anderson D C, Herzog C A, Qureshi A I. Clinical    epidemiology of atrial fibrillation and related cerebrovascular    events in the United States. Neurologist 2008; 14:143-50.-   27. Lip G Y, Kakar P, Watson T. Atrial fibrillation—the growing    epidemic.[comment]. Heart 2007; 93:542-3.-   28. Nademanee K, McKenzie J, Kosar E, et al. A new approach for    catheter ablation of atrial fibrillation: mapping of the    electrophysiologic substrate.[see comment]. Journal of the American    College of Cardiology 2004; 43:2044-53.-   29. Nademanee K, Schwab M C, Kosar E M, et al. Clinical outcomes of    catheter substrate ablation for high-risk patients with atrial    fibrillation. Journal of the American College of Cardiology 2008;    51:843-9-   30. Taylor G W, Kay G N, Zheng X, Bishop S, Ideker R E. Pathological    effects of extensive radiofrequency energy applications in the    pulmonary veins in dogs. Circulation 2000; 101:1736-42.-   31. Estner H L, Hessling G, Ndrepepa G, et al. Electrogram-guided    substrate ablation with or without pulmonary vein isolation in    patients with persistent atrial fibrillation. Europace 2008;    10:1281-7-   32. Weerasooriya R, Khairy P, Litalien J, et al. Catheter ablation    for atrial fibrillation: are results maintained at 5 years of    follow-up? J Am Coll Cardiol 2011; 57:160-6-   33. Ben Morrison T, Jared Bunch T, Gersh B J. Pathophysiology of    concomitant atrial fibrillation and heart failure: implications for    management. Nat Clin Pract Cardiovasc Med 2009; 6:46-56-   34. Nattel S. From guidelines to bench: implications of unresolved    clinical issues for basic investigations of atrial fibrillation    mechanisms. Can J Cardiol 2011; 27:19-26-   35. Nattel S, Burstein B, Dobrev D. Atrial remodeling and atrial    fibrillation: mechanisms and implications. Circ Arrhythm    Electrophysiol 2008; 1:62-73-   36. Youn J Y, Zhang J, Zhang Y, et al. Oxidative stress in atrial    fibrillation: an emerging role of NADPH oxidase. J Mol Cell Cardiol    2013; 62:72-9-   37. Maejima Y, Kuroda J, Matsushima S, Ago T, Sadoshima J.    Regulation of myocardial growth and death by NADPH oxidase. J Mol    Cell Cardiol 2011-   38. Kohlhaas M, Maack C. Interplay of defective    excitation-contraction coupling, energy starvation, and oxidative    stress in heart failure. Trends Cardiovasc Med 2011; 21:69-73-   39. Maulik S K, Kumar S. Oxidative stress and cardiac hypertrophy: a    review. Toxicol Mech Methods 2012; 22:359-66-   40. Hori M, Nishida K. Oxidative stress and left ventricular    remodelling after myocardial infarction. Cardiovascular Research    2009; 81:457-64-   41. Tsai K H, Wang W J, Lin C W, et al. NADPH oxidase-derived    superoxide anion-induced apoptosis is mediated via the JNK-dependent    activation of NF-kappaB in cardiomyocytes exposed to high glucose. J    Cell Physiol 2012; 227:1347-57-   42. Erickson J R, He B J, Grumbach I M, Anderson M E. CaMKII in the    cardiovascular system: sensing redox states. Physiol Rev 2011;    91:889-915-   43. Cave A C, Brewer A C, Narayanapanicker A, et al. NADPH oxidases    in cardiovascular health and disease. Antioxid Redox Signal 2006;    8:691-728-   44. Zhang P, Hou M, Li Y, et al. NADPH oxidase contributes to    coronary endothelial dysfunction in the failing heart. Am J Physiol    Heart Circ Physiol 2009; 296:H840-6-   45. Dworakowski R, Alom-Ruiz S P, Shah A M. NADPH oxidase-derived    reactive oxygen species in the regulation of endothelial phenotype.    Pharmacol Rep 2008; 60:21-8-   46. Zhang M, Brewer A C, Schroder K, et al. NADPH oxidase-4 mediates    protection against chronic load-induced stress in mouse hearts by    enhancing angiogenesis. Proc Natl Acad Sci USA 2010; 107:18121-6-   47. Huang C X, Liu Y, Xia W F, Tang Y H, Huang H. Oxidative stress:    a possible pathogenesis of atrial fibrillation. Med Hypotheses 2009;    72:466-7-   48. Carnes C A, Janssen P M, Ruehr M L, et al. Atrial glutathione    content, calcium current, and contractility. J Biol Chem 2007;    282:28063-73-   49. Carnes C A, Chung M K, Nakayama T, et al. Ascorbate attenuates    atrial pacing-induced peroxynitrite formation and electrical    remodeling and decreases the incidence of postoperative atrial    fibrillation. Circ Res 2001; 89:E32-8-   50. Kim Y M, Guzik T J, Zhang Y H, et al. A myocardial Nox2    containing NAD(P)H oxidase contributes to oxidative stress in human    atrial fibrillation. Circ Res 2005; 97:629-36.-   51. Cucoranu I, Clempus R, Dikalova A, et al. NAD(P)H Oxidase 4    Mediates Transforming Growth Factor-1-Induced Differentiation of    Cardiac Fibroblasts Into Myofibroblasts. Circulation Research 2005;    97:900-7.-   52. Zhang J, Youn J Y, Kim A, et al. NOX4-dependent Hydrogen    Peroxide Overproduction in Human Atrial Fibrillation and HL-1 Atrial    Cells: Relationship to Hypertension. Frontiers in Physiology 2012;    3.-   53. Shryock J C, Song Y, Rajamani S, Antzelevitch C, Belardinelli L.    The arrhythmogenic consequences of increasing late I_(Na) in the    cardiomyocyte. Cardiovasc Res 2013; 99:600-11-   54. Aistrup G L, Balke C W, Wasserstrom J A. Arrhythmia triggers in    heart failure: the smoking gun of [Ca2+]i dysregulation. Heart    Rhythm 2011; 8:1804-8.-   55. Antoons G, Sipido K R. Targeting calcium handling in    arrhythmias. Europace 2008; 10:1364-9.-   56. Laurita K R, Rosenbaum D S. Mechanisms and potential therapeutic    targets for ventricular arrhythmias associated with impaired cardiac    calcium cycling. J Mol Cell Cardiol 2008; 44:31-43-   57. Volders P G, Vos M A, Szabo B, et al. Progress in the    understanding of cardiac early afterdepolarizations and torsades de    pointes: time to revise current concepts. Cardiovasc Res 2000;    46:376-92.-   58. Laurita K R, Rosenbaum D S. Cellular mechanisms of    arrhythmogenic cardiac alternans. Prog Biophys Mol Biol 2008;    97:332-47-   59. Chou C C, Nihei M, Zhou S, et al. Intracellular calcium dynamics    and anisotropic reentry in isolated canine pulmonary veins and left    atrium. Circulation 2005; 111:2889-97-   60. Li D, Melnyk P, Feng J, et al. Effects of Experimental Heart    Failure on Atrial Cellular and Ionic Electrophysiology. Circulation    2000; 101:2631-8-   61. Yeh Y-H, Wakili R, Qi X-Y, et al. Calcium-Handling Abnormalities    Underlying Atrial Arrhythmogenesis and Contractile Dysfunction in    Dogs With Congestive Heart Failure. Circ Arrhythm Electrophysiol    2008; 1:93-102-   62. Kuster G M, Lancel S, Zhang J, et al. Redox-mediated reciprocal    regulation of SERCA and Na+-Ca2+ exchanger contributes to    sarcoplasmic reticulum Ca2+ depletion in cardiac myocytes. Free    Radical Biology and Medicine 2010; 48:1182-7-   63. Luo A, Ma J, Zhang P, Zhou H, Wang W. Sodium Channel Gating    Modes During Redox Reaction. Cellular Physiology and Biochemistry    2007; 19:9-20-   64. Valdivia C R, Chu W W, Pu J, et al. Increased late sodium    current in myocytes from a canine heart failure model and from    failing human heart. Journal of Molecular and Cellular Cardiology    2005; 38:475-83-   65. Song Y, Shryock J C, Belardinelli L. An increase of late sodium    current induces delayed afterdepolarizations and sustained triggered    activity in atrial myocytes. American Journal of Physiology—Heart    and Circulatory Physiology 2008; 294:H2031-H9-   66. Wasserstrom J A, Sharma R, O'Toole M J, et al. Ranolazine    Antagonizes the Effects of Increased Late Sodium Current on    Intracellular Calcium Cycling in Rat Isolated Intact Heart. Journal    of Pharmacology and Experimental Therapeutics 2009-   67. Undrovinas N, Maltsev V, Belardinelli L, Sabbah H, Undrovinas A.    Late sodium current contributes to diastolic cell    Ca&lt;sup&gt;2+&lt;/sup&gt; accumulation in chronic heart failure.    The Journal of Physiological Sciences 2010; 60:245-57-   68. Terentyev D, Gyorke I, Belevych A E, et al. Redox Modification    of Ryanodine Receptors Contributes to Sarcoplasmic Reticulum Ca2+    Leak in Chronic Heart Failure. Circulation Research 2008;    103:1466-72-   69. Gonzalez D R, Beigi F, Treuer A V, Hare J M. Deficient ryanodine    receptor S-nitrosylation increases sarcoplasmic reticulum calcium    leak and arrhythmogenesis in cardiomyocytes. Proc Natl Acad Sci USA    2007; 104:20612-7-   70. Marx S O, Marks A R. Dysfunctional ryanodine receptors in the    heart: new insights into complex cardiovascular diseases. J Mol Cell    Cardiol 2013; 58:225-31-   71. Bootman M D, Smyrnias I, Thul R, Coombes S, Roderick H L. Atrial    cardiomyocyte calcium signalling. Biochim Biophys Acta 2011;    1813:922-34-   72. Niggli E, Ullrich N D, Gutierrez D, Kyrychenko S, Polakova E,    Shirokova N. Posttranslational modifications of cardiac ryanodine    receptors: Ca(2+) signaling and E C-coupling. Biochim Biophys Acta    2013; 1833:866-75-   73. Donoso P, Sanchez G, Bull R, Hidalgo C. Modulation of cardiac    ryanodine receptor activity by ROS and RNS. Front Biosci (Landmark    Ed) 2011; 16:553-67-   74. Terentyev D, Gyorke I, Belevych A E, et al. Redox modification    of ryanodine receptors contributes to sarcoplasmic reticulum Ca2+    leak in chronic heart failure. Circ Res 2008; 103:1466-72-   75. Belevych A E, Terentyev D, Terentyeva R, et al. Shortened Ca2+    signaling refractoriness underlies cellular arrhythmogenesis in a    postinfarction model of sudden cardiac death. Circ Res 2012;    110:569-77.-   76. Swaminathan P D, Purohit A, Hund T J, Anderson M E.    Calmodulin-dependent protein kinase linking heart failure and    arrhythmias. Circ Res 2012; 110:1661-77-   77. Purohit A, Rokita A G, Guan X, et al. Oxidized    Ca2+/Calmodulin-Dependent Protein Kinase II Triggers Atrial    Fibrillation. Circulation 2013; 128:1748-57.-   78. Ashpole N M, Herren A W, Ginsburg K S, et al.    Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates    cardiac sodium channel NaV1.5 gating by multiple phosphorylation    sites. J Biol Chem 2012; 287:19856-69-   79. Hund T J, Koval O M, Li J, et al. A beta(I V)-spectrin/CaMKII    signaling complex is essential for membrane excitability in mice. J    Clin Invest 2010; 120:3508-19.-   80. Hashambhoy Y L, Winslow R L, Greenstein J L. CaMKII-dependent    activation of late I_(Na) contributes to cellular arrhythmia in a    model of the cardiac myocyte. Conf Proc IEEE Eng Med Biol Soc 2011;    2011:4665-8.-   81. Christensen M D, Dun W, Boyden P A, Anderson M E, Mohler P J,    Hund T J. Oxidized calmodulin kinase II regulates conduction    following myocardial infarction: a computational analysis. PLoS    Comput Biol 2009; 5:e1000583.-   82. Arora R. Recent insights into the role of the autonomic nervous    system in the creation of substrate for atrial fibrillation:    implications for therapies targeting the atrial autonomic nervous    system. Circ Arrhythm Electrophysiol 2012; 5:850-9-   83. Kong M H, Piccini J P, Bahnson T D. Efficacy of adjunctive    ablation of complex fractionated atrial electrograms and pulmonary    vein isolation for the treatment of atrial fibrillation: a    meta-analysis of randomized controlled trials. Europace 2011;    13:193-204.-   84. Kabra R, Singh J P. Catheter ablation targeting complex    fractionated atrial electrograms for the control of atrial    fibrillation. Curr Opin Cardiol 2012; 27:49-54-   85. Li D, Fareh S, Leung T K, Nattel S. Promotion of atrial    fibrillation by heart failure in dogs: atrial remodeling of a    different sort. Circulation 1999; 100:87-95.-   86. Koduri H, Ng J, Cokic I, et al. Contribution of fibrosis and the    autonomic nervous system to atrial fibrillation electrograms in    heart failure. Circ Arrhythm Electrophysiol 2012; 5:640-9-   87. Yeh Y H, Kuo C T, Chan T H, et al. Transforming growth    factor-beta and oxidative stress mediate tachycardia-induced    cellular remodelling in cultured atrial-derived myocytes. Cardiovasc    Res 2011; 91:62-70.-   88. Zhang J, Youn J Y, Kim A Y, et al. NOX4-Dependent Hydrogen    Peroxide Overproduction in Human Atrial Fibrillation and HL-1 Atrial    Cells: Relationship to Hypertension. Front Physiol 2012; 3:140.-   89. Vescovo G, Ravara B, Dalla Libera L. Skeletal muscle    myofibrillar protein oxidation and exercise capacity in heart    failure. Basic Res Cardiol 2008; 103:285-90-   90. Wasserstrom J A, Sharma R, Kapur S, et al. Multiple defects in    intracellular calcium cycling in whole failing rat heart. Circ Heart    Fail 2009; 2:223-32.-   91. Shinagawa K, Derakhchan K, Nattel S. Pharmacological prevention    of atrial tachycardia induced atrial remodeling as a potential    therapeutic strategy. Pacing Clin Electrophysiol 2003; 26:752-64-   92. Schotten U, Verheule S, Kirchhof P, Goette A. Pathophysiological    mechanisms of atrial fibrillation: a translational appraisal.    Physiol Rev 2011; 91:265-325.-   93. Arora R, Ng J, Ulphani J, et al. Unique autonomic profile of the    pulmonary veins and posterior left atrium. J Am Coll Cardiol 2007;    49:1340-8-   94. Arora R, Ulphani J S, Villuendas R, et al. Neural substrate for    atrial fibrillation: implications for targeted parasympathetic    blockade in the posterior left atrium. Am J Physiol Heart Circ    Physiol 2008; 294:H134-44-   95. Ng J, Villuendas R, Cokic I, et al. Autonomic remodeling in the    left atrium and pulmonary veins in heart failure: creation of a    dynamic substrate for atrial fibrillation. Circ Arrhythm    Electrophysiol 2011; 4:388-96-   96. Arora R, Verheule S, Scott L, et al. Arrhythmogenic substrate of    the pulmonary veins assessed by high-resolution optical mapping.    Circulation 2003; 107:1816-21-   97. Wasserstrom J A, Shiferaw Y, Chen W, et al. Variability in    timing of spontaneous calcium release in the intact rat heart is    determined by the time course of sarcoplasmic reticulum calcium    load. Circ Res 2010; 107:1117-26.-   98. Efimov I R, Nikolski V P, Salama G. Optical imaging of the    heart. Circ Res 2004; 95:21-33.-   99. Kong W, Ideker R E, Fast V G. Intramural optical mapping of V(m)    and Ca(i)2+ during long-duration ventricular fibrillation in canine    hearts. Am J Physiol Heart Circ Physiol 2012; 302:H1294-305-   100. Cutler M J, Plummer B N, Wan X et al. Aberrant S-nitrosylation    mediates calcium-triggered ventricular arrhythmia in the intact    heart. Proc Natl Acad Sci USA 2012; 109:18186-91.-   101. Gonzalez D R, Treuer A V, Castellanos J, Dulce R A, Hare J M.    Impaired S-nitrosylation of the ryanodine receptor caused by    xanthine oxidase activity contributes to calcium leak in heart    failure. J Biol Chem 2010; 285:28938-45-   102. Katra R P, Laurita K R. Cellular mechanism of calcium-mediated    triggered activity in the heart. Circ Res 2005; 96:535-42.-   103. Sridhar A, Nishijima Y, Terentyev D, et al. Chronic heart    failure and the substrate for atrial fibrillation. Cardiovascular    Research 2009; 84:227-36-   104. Dean D A. Nonviral gene transfer to skeletal, smooth, and    cardiac muscle in living animals. American journal of physiology    2005; 289:C233-45.-   105. Nattel S, Shiroshita-Takeshita A, Brundel B J, Rivard L.    Mechanisms of atrial fibrillation: lessons from animal models.    Progress in Cardiovascular Diseases 2005; 48:9-28-   106. Bovo E, Lipsius S L, Zima A V. Reactive oxygen species    contribute to the development of arrhythmogenic Ca(2)(+) waves    during beta-adrenergic receptor stimulation in rabbit    cardiomyocytes. J Physiol 2012; 590:3291-304.-   107. Ng J, Goldberger J J. Understanding and interpreting dominant    frequency analysis of A F electrograms. J Cardiovasc Electrophysiol    2007; 18:680-5-   108. Everett T H 4th M J, Kok L C, Akar J G, Haines D E. Assessment    of global atrial fibrillation organization to optimize timing of    atrial defibrillation. Circulation 2001; 103:2857-61.-   109. Lin Y J, Tai C T, Kao T, et al. Consistency of complex    fractionated atrial electrograms during atrial fibrillation. Heart    Rhythm 2008; 5:406-12-   110. Ng J, Borodyanskiy A I, Chang E T, et al. Measuring the    Complexity of Atrial Fibrillation Electrograms. Journal of    Cardiovascular Electrophysiology 2010; 21:649-55.-   111. Po S S, Scherlag B J, Yamanashi W S, et al. Experimental model    for paroxysmal atrial fibrillation arising at the pulmonary    vein-atrial junctions.[see comment]. Heart Rhythm 2006; 3:201-8-   112. Ng J, Kadish A H, Goldberger J J. Technical considerations for    dominant frequency analysis. J Cardiovasc Electrophysiol 2007;    18:757-64.-   113. Dibs S R, Ng J, Arora R, Passman R S, Kadish A H, Goldberger    J J. Spatiotemporal characterization of atrial activation in    persistent human atrial fibrillation: multisite electrogram analysis    and surface electrocardiographic correlations—a pilot study. Heart    Rhythm 2008; 5:686-93-   114. Scherr D, Dalal D, Cheema A, et al. Long- and short-term    temporal stability of complex fractionated atrial electrograms in    human left atrium during atrial fibrillation. J Cardiovasc    Electrophysiol 2009; 20:13-21.-   115. Lau D H, Maesen B, Zeemering S, Verheule S, Crijns H J,    Schotten U. Stability of complex fractionated atrial electrograms: a    systematic review. J Cardiovasc Electrophysiol 2012; 23:980-7.-   116. Lammers W J, Schalij M J, Kirchhof C J, Allessie M A.    Quantification of spatial inhomogeneity in conduction and initiation    of reentrant atrial arrhythmias. Am J Physiol 1990; 259:H1254-63.-   117. Koduri H, Ng J, Cokic I, et al. Contribution of fibrosis and    the autonomic nervous system to atrial fibrillation electrograms in    heart failure. Circ Arrhythm Electrophysiol 2012; 5:640-9.-   118. Ng J, Borodyanskiy A I, Chang E T, et al. Measuring the    complexity of atrial fibrillation electrograms. J Cardiovasc    Electrophysiol 2010; 21:649-55.

All patents, patent applications, patent application publications andother publications cited herein are hereby incorporated by reference asif set forth in their entirety.

It should be understood that the methods, procedures, operations,composition, and systems illustrated in figures may be modified withoutdeparting from the spirit of the present disclosure. For example, thesemethods, procedures, operations, devices and systems may comprise moreor fewer steps or components than appear herein, and these steps orcomponents may be combined with one another, in part or in whole.

Furthermore, the present disclosure is not to be limited in terms of theparticular embodiments described in this application, which are intendedas illustrations of various embodiments. Many modifications andvariations can be made without departing from its scope and spirit.Functionally equivalent methods and apparatuses within the scope of thedisclosure, in addition to those enumerated herein, will be apparent tothose skilled in the art from the foregoing descriptions.

The invention claimed is:
 1. A method of inhibiting oxidative stress ina subject having atrial or ventricular arrhythmias, ventricular failureor heart failure, comprising administering an effective amount of a NOX2inhibitor agent to the subject, wherein said administering is underconditions such that a level of oxidative stress in myocardial tissue isreduced or eliminated.
 2. The method of claim 1, wherein said arrhythmiacomprises atrial fibrillation.
 3. The method of claim 1, wherein theNOX2 shRNA is SEQ ID NO:
 1. 4. The method of claim 1, whereinadministering an effective amount of a NOX2 inhibitor agent to thesubject comprises: (a) providing an isolated therapeutic DNA comprisingan small hairpin RNA (shRNA) against NOX2 mRNA (NOX2 shRNA) expressionvector that encodes and expresses NOX2 shRNA in vivo; and (b)administering the isolated therapeutic DNA to myocardial tissue of thesubject.
 5. The method of claim 4, wherein the myocardial tissuecomprises at least one of atrial tissue or ventricle tissue.
 6. Themethod of claim 4, wherein administering the isolated therapeutic DNA tomyocardial tissue of the subject comprises injecting the isolatedtherapeutic DNA.
 7. The method of claim 1, further comprising assessinga parameter of atrial tissue status in the subject.
 8. The method ofclaim 7, wherein assessing a parameter of atrial tissue status in thesubject comprises monitoring an electrophysiological measurementassociated with AF or assessing fibrosis status for a region of themyocardial tissue after administering NOX2 inhibitor agent to thesubject.
 9. The method of claim 7, wherein assessing a parameter ofatrial tissue status in the subject comprises monitoring anelectrophysiological measurement associated with AF selected from AFonset, AF duration, conductivity and conductive inhomogeneity index. 10.A method of claim 2, further comprising assessing an AF characteristicfollowing administering the isolated therapeutic DNA.
 11. The method ofclaim 10, wherein the AF characteristic comprises at least one memberselected from a group consisting of AF duration, AF episodeinducibility, effective refractory periods and conduction inhomogeneityindex.
 12. The method of claim 1, further comprising assessing at leastone member selected from a group consisting of NOX2 nucleic acid, NOX2polypeptide, superoxide production and fibrosis.
 13. A method oftreating a subject having atrial or ventricular arrhythmias, ventricularfailure or heart failure, comprising administering an effective amountof a NOX2 inhibitor agent to the subject, wherein said administering isunder conditions such that a level of atrial or ventricular arrhythmiasis reduced or eliminated.
 14. The method of claim 13, wherein saidarrhythmia comprises atrial fibrillation.
 15. The method of claim 13,wherein administering an effective amount of a NOX2 inhibitor agent tothe subject (a) providing an isolated therapeutic nucleic acidcomprising a small hairpin RNA against NOX2 mRNA (NOX2 shRNA) in vivo;and (b) administering the isolated therapeutic nucleic acid tomyocardial tissue of the subject.
 16. The method of claim 15, furthercomprising: (c) assessing AF status for the myocardial tissue afteradministration of the therapeutic nucleic acid.
 17. The method of claim16, wherein the AF status comprises at least one member selected fromthe group consisting of AF duration, AF episode inducibility, effectiverefractory periods and conduction inhomogeneity index.
 18. The method ofclaim 13, further comprising assessing at least one member selected froma group consisting of NOX2 nucleic acid, NOX2 polypeptide, superoxideproduction and fibrosis.
 19. The method of claim 13, wherein theisolated therapeutic nucleic acid is expressed from an expression vectorencoding the NOX2 shRNA.
 20. A pharmaceutical composition for inhibitingoxidative stress in a subject having atrial or ventricular arrhythmias,ventricular failure or heart failure, comprising an isolated nucleicacid encoding a small hairpin RNA against NOX2 mRNA (NOX2 rhRNA).